Tocotrienols and tocotrienol-like compounds and methods for their use

ABSTRACT

The present invention relates to novel tocotrienols and tocotrienol-like compounds displaying biological activity. The tocotrienols and tocotrienol-like compounds of this invention may be conveniently obtained from biological sources or by chemical synthesis and may be used in pharmaceutical compositions, foodstuffs and dietary supplements. This invention also relates to the use of tocotrienols, tocotrienol-like compounds, and mixtures thereof, as hypocholesterolemic, antithrombotic, antioxidizing, antiatherogenic, antiinflammatory and immunoregulatory agents, or as agents useful to decrease lipoprotein (a) concentration in the blood or to increase feed conversion efficiency.

This application was filed under 35 U.S.C. §371 based on InternationalApplication No. PCT/US92/10277, filed Nov. 20, 1992, which in turn is acontinuation-in-part of U.S. application Ser. No. 07/796,486, filed Nov.22, 1991, now abandoned. All of the above applications are expresslyincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel tocotrienols and tocotrienol-likecompounds displaying biological activity. The tocotrienols andtocotrienol-like compounds of this invention may be convenientlyobtained from biological sources or by chemical synthesis and may beused in pharmaceutical compositions, foodstuffs and dietary supplements.This invention also relates to the use of tocotrienols, tocotrienol-likecompounds, and mixtures thereof, as hypocholesterolemic, antithrombotic,antioxidizing, antiatherogenic, antiinflammatory and immunoregulatoryagents, or as agents useful to decrease lipoprotein (a) concentration inthe blood or to increase feed conversion efficiency.

BACKGROUND OF THE INVENTION

Plant constituents have been proven useful in the prevention andtreatment of a wide variety of diseases and conditions. For example,barley has been shown to be particularly effective in lowering lipidlevels in animal models (A. A. Qureshi et al., "Suppression OfCholesterogenesis By Plant Constituents", Lipids, 20, pp. 817-24(1985)). More specifically, α-tocotrienol, a chromanol isolated frombarley extract, has been identified as a therapeutic agent forhypercholesterolemia (A. A. Qureshi et al., "The Structure Of AnInhibitor Of Cholesterol Biosynthesis Isolated From Barley", J. Biol.Chem., 261, pp. 10544-50 (1986)). In addition, tocotrienol,γ-tocotrienol and δ-tocotrienol have also been shown to reducehypercholesterolemia in mammals (European patent application 421,419).

Hypercholesterolemia involves high serum cholesterol levels and is acausative agent of diseases including arteriosclerosis, atherosclerosis,cardiovascular disease and xanthomatosis. In addition, high serumcholesterol levels are seen in patients suffering from diseasesincluding diabetes mellitus, familial hypercholesterolemia, acuteintermittent prothyria, anorexia nervosa, nephrotic syndrome, primarycirrhosis and various liver disorders, such as hepatitis and obstructivejaundice. Improvement of lipoprotein profiles and a decrease in totalserum and low density lipoprotein cholesterol have been shown to retardthe progression of such diseases, as well as to induce regression ofclinically significant lesions in hypercholesterolemic patients.

Although the relationship between hypercholesterolemia and its manyassociated diseases, most notably cardiovascular disease, has beenextensively studied, no clear answer to this world-wide problem has yetbeen found. As a result, coronary artery disease remains the leadingcause of death in the United States and other developed countries.Coronary artery disease is the result of complex interactions between alarge number of different processes, including lipoprotein metabolism,aggregation of blood platelets, blood coagulation and fibrinolysis.Accordingly, the cardiovascular risk profile of a given patient isdependent on these interactions.

In addition to lowering cholesterol levels, the cardiovascular riskprofile of a patient may also be reduced by decreasing the levels ofother factors in the serum and the blood. For example, reduction ofthromboxane A₂ generation (measured by the levels of thromboxane B₂, astable metabolite of thromboxane A₂) and platelet factor 4 levels in theserum lessens the risk of cardiovascular disease because of decreasedthrombogenic activity.

Thromboxane A₂ and platelet factor 4 levels are also associated withother biological activities. For example, when reduction of thesefactors is accompanied by a reduction in macrophage cell count, lowertumor necrosis factor (TNF) levels and lower arachidonic acid levels inbodily tissues, reduced levels of prostaglandins, leukotrienes andinterleukins are implicated. Reduction of these factors, therefore,leads to a decrease in the inflammation accompanying a wide variety ofdiseases. In addition, since prostaglandins inhibit glucose-inducedinsulin release and increase glucagon secretion, an increased insulin toglucagon ratio may also result from the reduction in prostaglandins.Such an increase is useful in improving glucose intolerance in diabetesmellitus and restoration of acute glucose-induced insulin response innon-insulin-dependent diabetes mellitus.

It has been noted that there is a low incidence of cardiovasculardisease in populations consuming large amounts of cereal grains. Solubleand insoluble fibers have, in the past, been viewed as the agentsresponsible for cholesterol reduction in such populations (see D.Kritchevsky et al., "Fiber, Hypercholesterolemia and Atherosclerosis",Lipids 13, pp. 366-69 (1978)). Recently, the hypocholesterolemic effectsof cereal grains have been attributed to natural components of thegrains--tocotrienols ("T₃ ") and structurally similar compounds, such astocopherols ("T"). Tocotrienols and tocopherols occur naturally in smallquantities in a wide variety of plant sources, such as rice bran, palmoil and barley (A. A. Qureshi et al., "Lowering of Serum Cholesterol inHypercholesterolemic Humans by Tocotrienols (Palmvitee)", Am. J. Clin.Nutr., 53, pp. 1021S-6S (1991)).

As a class, the tocopherols, including d-α-tocopherol (vitamin E), havebeen extensively studied. As a result of these studies, certainbiological activities have been attributed to the tocopherols. Suchactivities include platelet aggregation and antioxidant functions (see,for example, E. Niki et al., "Inhibition of Oxidation of Biomembranes ByTocopherol", Annals of the New York Academy of Sciences, 570, pp. 23-31(1989) and K. Fukuzawa et al., "Increased Platelet-Activating Factor(PAF) Synthesis in Polymorphonuclear Leukocytes of Vitamin E-DeficientRats", Annals of the New York Academy of Sciences, 570, pp. 449-453(1989)). Although the exact structure-function relationship is notknown, several experiments have highlighted the importance of the phytylside chain in the biological activity of tocopherols (see W. A. Skinneret al., "Antioxidant Properties of α-Tocopherol Derivatives andRelationships of Antioxidant Activity to Biological Activity", Lipids,5(2), pp. 184-186 (1969) and A. T. Diplock, "Relationship of TocopherolStructure to Biological Activity, Tissue Uptake, and ProstaglandinBiosynthesis", Annals of the New York Academy of Sciences, 570, pp.73-84 (1989)).

In contrast to the tocopherols, interest in the tocotrienols has beenlimited, as those compounds were not typically considered to bebiologically useful. Recently, however, studies have indicated thattocotrienols may be biologically active. For example, U.S. Pat. No.4,603,142 identifies d-α-tocotrienol, isolated from barley extracts, asan inhibitor of cholesterol biosynthesis. See also A. A. Qureshi et al.(1986), Supra. Various human and animal studies have confirmed theimpact of pure tocotrienols, isolated from barley, oats and palm oil, oncholesterol biosynthesis, specifically LDL-cholesterol (A. A. Qureshi etal., "Dietary Tocotrienols Reduce Concentrations of Plasma Cholesterol,Apolipoprotein B, Thromboxane B₂ and Platelet Factor 4 In Pigs WithInherited Hyperlipidemias", Am. J. Chin. Nutr., pp. 1042S-46S (1991); A.A. Qureshi et al., "Lowering Of Serum Cholesterol InHypercholesterolemic Humans By Tocotrienols (Palmvitee)", Am. J. Clin.Nutro, 53, pp. 1021S-26S (1991); D.T.S. Tan et al , "The Effect Of PalmOil Vitamin E Concentrate On The Serum And Lipoprotein Lipids InHumans", Am. J. Clin. Nutr., 53, pp. 1027S-30S (1991)). In addition,tocotrienol, γ-and δ-tocotrienol have been indicated for use in thetreatment of hypercholesterolemia, hyperlipidemia and thromboembolicdisorders (European patent application 421,419).

The five known naturally occurring tocotrienols have been designatedtocotrienol, α-, β-, γ- and δ-tocotrienol. Those compounds exhibitvarying degrees of hypercholesterolemic activity and have also been usedas antithrombotic agents and antioxidants. α-T₃, for example, displaysantioxidant activity against lipid peroxidation in rat liver microsomalmembranes and against oxidative damage of cytochrome P-450 (E.Serbinova, Free Radical Biology and Medicine, in press (1991)). Despitethese activities, the known tocotrienols have not found wide-spreadtherapeutic use.

Accordingly, the need still exists for compounds which, as singleagents, can safely and effectively act as hypercholesterolemic,antithrombotic, antioxidizing, antiatherogenic, antiinflammatory andimmunoregulatory agents.

SUMMARY OF THE INVENTION

The present invention solves the problems referred to above by providingnovel tocotrienols, tocotrienol-like compounds, and mixtures thereof,that are useful as hypocholesterolemic, antithrombotic, antioxidizing,antiatherogenic, antiinflammatory and immunoregulatory agents. Thesenovel tocotrienols and tocotrienol-like compounds are also useful indecreasing lipoprotein (a) ("Lp(a)") concentration in the blood,increasing feed conversion efficiency, and in the treatment orprevention of conditions such as fever, edema, diabetes mellitus,cancer, signs of aging, pain, rheumatoid diseases, septic shock, chronicfatigue syndrome and functio laesa.

The novel tocotrienols and tocotrienol-like compounds of the presentinvention are useful in inhibiting the synthesis of HMG-CoA reductase,reducing lipogenesis and increasing the HDL/LDL cholesterol ratio, aswell as reducing total serum cholesterol, low densitylipoprotein-cholesterol, Lp(a), apolipoprotein B, thromboxane A₂,platelet factor 4, triglycerides and glucose. Because of such individualactivities, these compounds are useful in treating diseases, forexample, coronary artery disease, which result from the interaction ofprocesses such as lipoprotein metabolism, aggregation of bloodplatelets, blood coagulation and fibrinolysis. Accordingly, thecompounds of this invention are useful to improve the overallcardiovascular profile of patients.

This invention also provides novel uses for the known tocotrienols asantiinflammatory and immunoregulatory agents, to decrease Lp(a)concentration in the blood, to increase feed conversion efficiency, andfor the treatment or prevention of conditions such as fever, edema,diabetes mellitus, pain, septic shock, chronic fatigue syndrome andfunctio laesa.

Advantageously, the tocotrienol and tocotrienol-like compounds reduceserum TNF and IL-1 levels. These activities render the compounds usefulin reducing active and chronic inflammation, such as that associatedwith rheumatoid disease. Also, such activities render the compoundsuseful in treating, preventing or lessening the severity ofimmunoregulatory diseases, such as autoimmune diseases, and inpreventing or treating pain, septic shock, chronic fatigue syndrome,functio laesa and oxidative conditions.

The effects of tocotrienol and tocotrienol-like compounds on lipidmetabolism also influence the regulation of antibody production. Thus,these compounds are useful in modulating immune function throughadjustments in fatty acid levels.

Tocotrienol and tocotrienol-like compounds also decrease glucose levelsby mediating the levels of insulin and glucagon. By increasing theinsulin to glucagon ratio in the blood, these compounds are useful inincreasing glucose intolerance in diabetes mellitus and restoring acuteglucose-induced insulin response in non-insulin dependent diabetesmellitus patients.

The novel tocotrienols and tocotrienol-like compounds of this inventionare characterized by specific structural characteristics and specificbiological activity or, alternatively, by specific high performanceliquid chromatography ("HPLC") elution profiles and specific biologicalactivity. More particularly, the compounds of this invention may becharacterized by three structural features: (1) a hydrogen donor group(or a group which can be hydrolyzed to a hydrogen donor group) attachedto an aromatic ring system, (2) an atom having at least one lone pair ofelectrons, said electrons being in conjugation with the aromatic systemand (3) a side chain comprising one or more isoprenoid orisoprenoid-like units attached to a position adjacent to that atom. Thisinvention also encompasses the hydrolysis and oxidation productsobtained from such compounds. In addition, the compounds of thisinvention having the above-mentioned structural characteristics are alsocharacterized by the ability to inhibit the activity ofβ-hydroxy-β-methyl glutaryl coenzyme A (HMG-CoA) reductase. Furthermore,these compounds are effective in the treatment or prevention of one ormore of the following diseases or conditions: hypercholesterolemicdiseases, thrombotic diseases, oxidative conditions, inflammation orimmunoregulatory diseases or, alternatively, in increasing feedconversion efficiency.

According to the alternate embodiment, the novel tocotrienols andtocotrienol-like compounds of this invention may be characterized by anelution time of at least 22 minutes under the following HPLC conditions:μ-Porasil column (Waters column, 10μ, 4 mm ×30 cm) using an isocraticsystem of hexane and isopropanol (99.75%:0.25%, v/v) at a flow rate of1.3 ml/min. The particular compound is detected at an exitationwavelength of 295 nm and an emission wavelength of 330 nm (fluorescencedetector) and UV absorption at 295 nm. In addition to the above-definedelution profile, the novel tocotrienols of this invention are alsocharacterized by the ability to inhibit the activity of HMG-CoAreductase.

According to this invention, tocotrienols and tocotrienol-like compoundsmay be conveniently obtained from biological sources. Alternatively,they may be synthesized using conventional chemical methodologies. In apreferred embodiment of this invention, the tocotrienols andtocotrienol-like compounds are isolated and purified from stabilizedbiological sources. This invention also encompasses the techniques usedto purify such compounds.

In a preferred embodiment of this invention, the tocotrienols,tocotrienol-like compounds and mixtures thereof, may be administered toan animal or a human as a pharmaceutical composition, a foodstuff or adietary supplement, to treat or prevent hypercholesterolemic diseases,thrombotic diseases, oxidative or atherogenic conditions, inflammationor immunoregulatory diseases. Advantageously, these compositions,foodstuffs and dietary supplements may also be used to increase feedconversion efficiency, decrease Lp(a) concentration in the blood and inthe treatment or prevention of fever, edema, diabetes mellitus, cancer,signs of aging, pain, rheumatoid diseases, septic shock, chronic fatiguesyndrome and functio laesa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 depict the effects of different tocotrienols from rice branoil on fatty acid concentrations in various tissues ofhypercholesterolemic swine.

FIG. 5 depicts the effects of GT301 (γ-T₃) on superoxide production inhuman peripheral blood neutrophils.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following terms are employed:

Biological source--Any natural or recombinant plant source, natural ortransgenic animal source, microbial source (e.g., bacteria), fungi,yeast, algae, higher plant source, or derivative thereof, which containsone or more tocopherol, tocotrienol or tocotrienol-like compounds.

Stabilization--A process effective to increase the recoverable amountsof tocopherols, tocotrienols and tocotrienol-like compounds in abiological source by one or a combination of: (1) inactivating enzymeswhich are capable of degrading tocotrienols and tocotrienol-likecompounds in that biological source, (2) breaking bonds or otherwiseinterfering with the interactions--such as hydrogen bonds, covalentbonds, ionic bonds, Van der Waals forces, London forces and hydrophobicor hydrophilic interactions which bind the desired products to proteins,sugars, lipids, carbohydrates, glycoproteins, other biological molecules(i.e., amino acids or nucleotides) or other membrane components, orcombinations thereof, in the biological source--which retaintocotrienols and tocotrienol-like compounds in that biological source,thus facilitating the release of those desired compounds, or (3)increasing the solubility of the tocotrienols and tocotrienol-likecompounds of that biological source beyond that prior to stabilizationor beyond the level of solubility of the tocotrienols of a correspondingnon-stabilized biological source. As a result of stabilization,tocotrienols and tocotrienol-like compounds in a biological source maybe recovered in higher yields than those realized from a correspondingnon-stabilized biological source.

Tocol--A mixture of one or more compounds selected from tocopherols (T),tocotrienols (T₃), and tocotrienol-like (T₃ -like) compounds.

Tocotrienol-like--Any biologically active compound which is contained inor derived from a biological source and (1) which is released, or whoserelease is facilitated, upon stabilizing that source or (2) whoserecoverable amount in that source is increased by stabilizing thatsource. Such tocotrienol-like compounds include any biologically activecompound displaying the biological activity of a tocotrienol whichinhibits the activity of HMG-CoA reductase as measured by an in vitroHMG-CoA reductase assay, such as that described in D. J. Shapiro et al.,"Microassay for β-hydroxy-β-methylglutaryl-CoA Reductase in Rat Liverand in L-cell Fibroblasts", Biochim. Biophys. Acta, 370, p. 369 (1974).Tocotrienol-like compounds include, but are not limited to, any electrontransfer ring compounds, anti-oxidant type compounds, redox compoundsand compounds similar to or containing the three structural featuresthat characterize the tocotrienols of this invention. Specific examplesof T₃ -like compounds are ubiquinones, plastoquinones, isoquinones,phylloquinones, benzoquinones, flavanols, flavanoids, coumarins,unsaturated terpenoids and unsaturated isoprenoids. The term "T₃ -likecompound" also encompasses analogues, homologs, isomers and derivativesof such compounds, such as prenylated derivatives or pyrolyticderivatives.

Preferred tocotrienol-like compounds have the following structures:##STR1## and salts, oxidation products and hydrolysis products thereofwherein:

R₁ and R₃ is each independently selected from the group consisting of H,halogen, OH, OCH₃ and C₁ -C₆ branched or unbranched alkyls;

R₂ is selected from the group consisting of OH, NHR₈, CO₂ Y, C(R₈)₂ CO₂H, and C₁ -C₈ branched or unbranched alkyls substituted by OH, NHR₈, CO₂H or C(R₈)₂ CO₂ H;

R₄ is selected from the group consisting of O, NH, CH--R₉, C═O andCH--OH;

R₅ is selected from the group consisting of CH₂, CHOH, O, S and NH:

R₆ is selected from the group consisting of H and C₁ -C₆ branched orunbranched alkyls;

Each R₇ independently represents ##STR2##

wherein R₁₀ is selected from the group consisting of H, NH₂ and C₁ -C₆branched or unbranched alkyls;

R₈ and R₉ is each independently selected from the group consisting of Hand C₁ -C₆ branched or unbranched alkyls;

Each R₁₁ is independently selected from the group consisting of H, C₁-C₆ branched or unbranched alkyls, CH₂ OH, CO₂ H or OH;

Y is H or C₁ -C₁₈ branched or unbranched alkyls, preferably C₁ -C₄alkyls;

Z is H, halogen, OH, CH₂ OH, CH₃, OCH₃ or COCH₃ ;

n is an integer from 0-4; and

m is an integer from 1-30, more preferably 1-20 or most preferably 3-10.

TRF--A tocotrienol-rich fraction obtained by the stabilization andextraction of a biological source. It typically contains varying amountsof the five known tocotrienols, the known tocopherols and the noveltocotrienols and tocotrienol-like compounds of this invention. Mostcommonly, the TRF will be composed of about 50% to about 90%tocotrienols and tocotrienol-like compounds. The TRF, may be used forany of the uses described herein for either the known tocotrienols orthe novel tocotrienols and tocotrienol-like compounds of this invention.

TRF Standard--A tocotrienol-rich fraction (TRF) obtained from palm oil(A. A. Qureshi et al. (1991), supra). The TRF Standard contains varyingamounts of α-, γ- and δ-tocotrienol and α-tocopherol but essentiallynone of the novel tocotrienol and tocotrienol-like compounds of thisinvention.

Enhanced--The state of a stabilized biological source, wherein therecoverable amount of the novel tocotrienols and tocotrienol-likecompounds of this invention is increased beyond that recoverable fromthe biological source prior to stabilization.

Foodstuff--Substances that can be used or prepared for use as food foran animal or a human. Foodstuffs include substances that may be used inthe preparation of food (such as frying oils) or food additives. Forexample, foodstuffs include animals used for human consumption or anyproduct therefrom, such as, for example, eggs. Such animals themselvesmay have ingested or been treated with one or more tocotrienol ortocotrienol-like compound, or foodstuffs containing them. As discussedherein, after ingesting or being otherwise administered tocotrienols ortocotrienol-like compounds, such animals retain the advantageoushypocholesterolemic, antithrombotic, antioxidizing, antiatherogenic,antiinflammatory and immunoregulatory properties of the tocotrienols andtocotrienol-like compounds.

HPLC elution time or profile--Except where otherwise noted, the term"HPLC elution time or profile" refers to the time necessary for a givencompound to elute from the HPLC column or the characteristic profile ofa mixture of compounds (20 μl ) under the following conditions:μ-Porasil column (Waters column, 10μ, 4 mm×30 cm) using an isocraticsystem of hexane and isopropanol (99.75%:0.25%, v/v) at a flow rate of1.3 ml/min. The compound is detected at an exitation wavelength of 295nm and an emission wavelength of 330 nm (fluorescence detector) and UVabsorption at 295 nm.

The individual tocotrienols and tocotrienol-like compounds of thisinvention constitute a novel class of biologically active compounds.Several of these compounds occur naturally in small quantities inbiological sources. Therefore, in a preferred embodiment of thisinvention, tocotrienols and tocotrienol-like compounds are recoveredfrom biological materials which have been stabilized and extractedaccording to the processes described in copending International patentapplication PCT/US91/03626, filed on May 23, 1991. The stabilization andrecovery process disclosed therein enhances or increases the yields oftocotrienols and tocotrienol-like compounds recovered from biologicalsources.

The tocotrienol and tocotrienol-like compounds may be recovered from anybiological materials including, but not limited to, oats, wheat, rye,barley, soybean, wheat germ, wheat bran, corn, rice (including wholekernel, husk or hull, endosperm and germ), cottonseed, milkweed, flax,sesame, rice bran, parboiled brown rice, brown rice flour, olives,vegetable oil distillant, fruit concentrate evaporate, barley bran, palmoil, wheat germ oil, rice bran oil, barley oil, coconut oil, cottonseedoil, soybean oil, other cereal grains and other cereal grain oils, planttissues, flowers, bushes (such as juniper), trees (such as pine andrubber), fruits (such as melons, berries, tomatoes and citrus fruits),vegetables, grasses (such as alfalfa), fungi (such as mushrooms),leaves, seeds (such as sesame, millet and pine), such as sesame seedsand pine seeds, stems, bark, roots, nuts (such as cashews and almonds)and legumes (such as peanuts), or portions thereof. We have noted thatthe tocotrienols and tocotrienol-like compounds decompose in thebiological source over time. Therefore, we prefer to use freshlyharvested biological sources. Most preferably, the biological source isfreshly harvested, stabilized rice bran.

Alternatively, the tocotrienols and tocotrienol-like compounds may beobtained from a non-stabilized source or synthesized according to knownchemical methodology. Typical synthetic routes are described in J. W.Scott et al., "Synthesis of (2R, 4'R, 8'R)-α-Tocopherol and (2R, 3'E,7'E)-α-Tocotrienol", Helv. Chem. Acta, 59, pp. 290-306 (1976) and P.Schudel et al., "Die Synthese yon rac. all-trans δ- und ε-Tocopherol",Helv Chem Acta., 46, pp 2517-2526 (1963). Am alternative syntheticscheme is proposed in European patent application 421,419.

The tocotrienols and tocotrienol-like compounds of this invention may bealtered by known chemical means to produce various derivatives oranalogues. Such derivatives or analogues may be more easily isolated inpure form, or more resistant to degradation, or possess other desiredcharacteristics. Such derivatives and analogues are also envisioned bythis invention. Known chemical means to alter the compounds of thisinvention include, but are not limited to, heating in the presence orabsence of air or in an inert or reactive gas environment (or in amixture of inert and active gases) and pyrolysis.

This invention encompasses the d- or l-isomer and the d, l-racemicmixture of each tocotrienol and tocotrienol-like compound. However, thenaturally occurring d-isomer is preferred. This invention also includesmixtures of at least one novel tocotrienol or tocotrienol-like compoundof this invention with one or more of the known tocotrienols.

The tocotrienols and tocotrienol-like compounds of this invention may becharacterized either by specific structural characteristics oralternatively, by a specific elution profile and specific biologicalactivity. In the former case, the compounds of this invention sharethree structural features: (1) a hydrogen donor group (or a group whichcan be hydrolyzed to a hydrogen donor group) attached to an aromaticring system, (2) an atom having at least one lone pair of electrons,said electrons being in conjugation with the aromatic system and (3) aside chain comprising one or more isoprenoid or isoprenoid-like unitsattached to a position adjacent to that atom. This invention alsoencompasses the hydrolysis and oxidation products obtained from suchcompounds. These compounds are effective in increasing feed conversionefficiency, decreasing Lp(a) concentration in the blood or,alternatively, in the treatment or prevention of one or more of thefollowing diseases or conditions: immunoregulatory disease,inflammation, fever, edema, diabetes mellitus, cancer, signs of aging,pain, rheumatoid diseases, septic shock, chronic fatigue syndrome andfunctic laesa.

According to an alternate embodiment, the novel tocotrienols adtocotrienol-like compounds of this invention may be characterized by anelution time and specific biological activity. These compounds eluteafter at least 22 minutes under the following high performance liquidchromatography (HPLC) conditions: Porasil column (Waters column, 10μ, 4mm×30 cm) using an isocratic system of hexane and isopropanol(99.75%:0.25%, v/v) at a flow rate of 1.3 ml/min. The compound isdetected at an exitation wavelength of 295 nm and an emission wavelengthof 330 nm (fluorescence detector) and UV absorption at 295 rn. Inaddition to the above-defined elution profile, the novel tocotrienolsand tocotrienol-like compounds of this invention are also characterizedby the ability to inhibit the activity of β-hydroxy-β-methyl glutarylcoenzyme A reductase ("HMG-CoA reductase").

The preferred class of compounds of this invention is of formula I:##STR3## above. As herein described above, the compounds of formula I donot include desmethyl-tocotrienol(3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3' (E), 7' (E),11'-trienyl)-2H-1-benzopyran-6-ol).*

Typical hydrolysis products of the compounds of formula I which are alsoenvisioned by this invention include, but are not limited to, compoundsof formula II: ##STR4## wherein R₁ -R₁₀, Z, n and m are as defined asabove. Preferred compounds are the compounds of formula I, wherein R₁,R₃ and Z are each independently H, halogen, OH, OCH₃ or CH₃ ; R is OH,OCH₃ or NH₂ ; R₄ is C═O, CH₂ or NH; R₅ is O, S, CH₂ or NH; R₆ is H orCH₃ ; R₁₀ and R₁₁ are each independently H or CH₃ ; n is 0 or 1; and mis 3-10.

The most preferred compounds are the compounds of formula I, wherein R₁,R₃ Z, R₈ and R₉ are each hydrogen, R₂ is OH, R₄ is CH₂, R₅ is CH₂ or O,R₆ is H or CH₃, R₁₀ is H or CH₃, n is 1 and m is 3.

The novel compound, 3,4-dihydro-2-(4,8,12-trimehtyltrideca-3' (E), 7'(E), 11' trienyl)-2H-1-benzopyran-6-ol ("P₂₅ "), is representative ofthe class of compounds according to formula I. The structure of thiscompound is shown below: ##STR5##

Tocotrienols and tocotrienol-like compounds of this invention having theabove-defined structural features are also characterized by biologicalactivity. They are useful in increasing feed conversion efficiency,decreasing Lp(a) concentration in the blood or in the treatment orprevention of one or more of the following diseases or conditions:immunoregulatory disease, inflammation, fever, edema, diabetes mellitus,cancer, signs of aging, pain, rheumatoid diseases, septic shock, chronicfatigue syndrome and functio laesa. They may also reduce the levels ofTNF, IL-1 and IL-1 stimmulatory products including possibly IL-2, IL-6,IL-8, GMCSF, prostaglandins and gamma interferon, while increasingantibody titers in response to foreign proteins. By virtue of theseactivities, these compounds may result in a decrease in the release ofsuperoxide and other cytotoxins produced by neutrophils, mast cells,basophils, monocyctes and macrophages, eosinophils, platelets,lymphocytes and polymorphonuclear leukocytes, endothelial tissue andother immunoregulatory tissues. These compounds may also effect anincrease in antibody titers.

HMG-CoA reductase catalyzes the rate-limiting step of cholesterolbiosynthesis. Therefore, a reduction in its activity decreases the totalamount of cholesterol in the serum of animals and humans alone, or incombination with a low fat, low cholesterol diet. The effects are mostnoticeable in hypercholesterolemic individuals with poor dietaryregimens. Typically, the tocotrienols and tocotrienol-like compounds ofthis invention reduce the activity of HMG-CoA reductase by at leastabout 15% in the in vitro assay described in Shapiro (supra), at aconcentration of 10-20 μg/ml. More preferably, however, the HMG-CoAreductase activity is reduced by at least about 20% and most preferablyby about 25%.

While not wishing to be bound by theory, we believe that some of theantiinflammatory, antioxidizing and immunoregulatory properties of thetocotrienol and tocotrienol-like compounds is a result of an inhibitionin the production of free arachidonic acid.

We believe that the inhibition in the production of arachidonic acid iscaused by either the inhibition of phospholipase A₂ or alternatively, byincreasing the amount of corticosterone level in the blood.Phospholipase A₂ cleaves at C-2 of phosphate head groups, resulting inthe release of free arachidonic acid. Arachidonic acid can then beconverted into a variety of biologically important molecules, such asthe prostaglandins and thromboxanes (via the cyclooxygenase pathway) andthe leukotrienes (via the lipoxygenase pathway). We believe that thehydrogen donor group of the tocotrienol and tocotrienol-like compoundsbonds with the phosphate or phospholipid groups of biological membranes.In conjunction with the aromatic system, it also plays a role in radicalscavenging. The isoprenoid tail may act to stabilize themembrane--possibly by forming weak bonds with the lipid side chains. Inaddition, by optionally replacing the tocotrienol methyl with othersubstituents (such as, for example, hydrogen) at position 2 on thechromanol ring, we are able to maximize the stabilization of themembrane. We believe that the aromatic system may also aid in tighteningthe surface of the membrane, thus increasing the steric hindrance forphospholipase A₂ and phospholipase C and decreasing its permeability.The aromatic system may also serve electron-transport and antioxidizingfunctions. Such a hypothesis would contribute to the explanation of thebroad spectrum of biological functions that are effected by theadministration of the tocotrienols and tocotrienol-like compoundsaccording to this invention.

This invention also includes several novel uses for known tocotrienols,as well as tocotrienols and tocotrienol compounds as described herein.Examples include their use as antiinflammatory, antiatherogenic andimmunoregulatory agents, for the treatment of fever, edema, diabetesmellitus, cancer, signs of aging, pain, septic shock, chronic fatiguesyndrome and functio laesa. In addition, tocotrienols andtocotrienol-like compounds are useful for increasing feed conversionefficiency--resulting in conversion of a greater percentage of food intoprotein rather than fat--and decreasing Lp(a) concentration in theblood. Known tocotrienols exhibit much of the same biological activityattributed to the novel tocotrienols and tocotrienol-like compounds ofthis invention. For example, known tocotrienols are capable of reducingtotal serum and LDL-cholesterol, apolipoprotein B, thromboxane A₂,platelet factor 4, triglycerides and glucose and inhibiting the activityof HMG-CoA reductase. They also reduce the levels of TNF and IL-1, andpossibly IL-2 and gamma interferon, while increasing antibody titers inresponse to foreign proteins. In combination, these factors may producea variety of advantageous and novel biological results, including adecrease in the release of superoxide and other cytotoxins produced byimmunoregulatory cells and an increase in antibody titers.

Tocotrienols and tocotrienol-like compounds according to this inventionare preferably recovered from stabilized biological sources.Alternatively, tocotrienols and tocotrienol-like compounds may berecovered from biological sources subjected to conventional foodprocessing or preparation techniques. The stabilization process,however, greatly increases the recovered yields of these compounds.Without wishing to be bound by theory, we believe that a significantamount of tocotrienols and tocotrienol-like compounds are, in nature,bound to proteins or linked to phosphate or phospholipid groups in themembranes of biological sources. The significant increase in recoveredamounts of these compounds realized by stabilization may be attributedto their release from protein or cleavage of phosphate or pyrophosphatemoieties attached to the hydroxy group of the benzene ring in thetocotrienol or the hydrogen donor group of the tocotrienol-likecompound. The application of heat and, optionally, pressureadvantageously releases the compounds of this invention so that they maybe recovered in good yield. Preferably, a combination of heat andpressure are used. Depending on the method of recovery used, thecompounds of this invention may be extracted together with othercomponents of the TRF.

We have found that microwaving is particularly effective to releasemembrane-bound T₃ and T3-like compounds. We have also found that thefollowing protocol maximizes the desired stabilization results: Anamount (typically from 1 g to 1 kg) of a ground biological source(preferably rice or rice bran) is placed in a pyrex dish (typically, 10cm-20 cm in diameter and 1.5 cm-15 cm in height). The dish can becovered or uncovered and optionally, a second dish containing a groundbiological source can be stacked above the first. We prefer using onecovered dish at a time. The biological source is then heated in amicrowave oven at the maximum level (preferably 600-1500 watts) for 1-5minute intervals, optionally in an ambient, vacuum or nitrogen blanketenvironment. Pressures of greater than 1 atmosphere may also optionallybe used. After each heating interval, the sample should be stirred tohomogeneity. The heating and stirring process is repeated 1-10 times. Weprefer repeating the process 3-5 time for a 0.5 kg sample. The TRF maythen be extracted, as described herein.

The TRF may be used directly, or it may be further separated in to itscomponent compounds. It may be desired to isolate the novel tocotrienolsand tocotrienol-like compounds from the TRF mixture.

The TRF typically contains varying degrees of each of the knowntocotrienols, plus additional Tocol products. Theoretically, each noveltocotrienol and tocotrienol-like compound should be separable from theknown tocopherols and tocotrienols in the TRF using standard silica gelHPLC methodology. However, in the case of rice bran, a complex mixtureresults. This resultant mixture contained3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E),7'(E),11'-trienyl)-2H-1-benzopyran-6-0l), a known tocotrienol that eluted by HPLC after about 21 minutes underthe specified conditions. For that reason, we refer to this compound as"P₂₁ ". In addition, the mixture contained3,4-dihydro-2-(4,8,12-trimethyltrideca-3' (E), 7' (E), 11'trienyl)-2H-1-benzopyran-6-ol), or "P₂₅ ", a novel tocotrienol thateluted by HPLC after about 25 minutes. And the mixture contained anapparent sterol which had a UV absorption maximum at 315 nm that elutedby HPLC after about 20 minutes ("P₂₀ "). These three compounds cannot beseparated using standard silica gel HPLC techniques.

Accordingly, the present invention includes novel techniques to purifytocotrienols and tocotrienol-like compounds from contaminants thatcannot be removed using a silica column. These techniques are especiallywell suited for separating tocopherols and tocotrienol-like compoundsfrom sterol and other waxy contaminants. In one process, the TRF isdissolved in an appropriate solvent, preferably hexane, and then boundto an amine or cyano column (1 ml). We prefer to use a Bond Elute amineor cyano column. The tocotrienols and tocotrienol-like compounds of thisinvention may then be selectively eluted from the column by anappropriate solvent system. Preferably, this solvent system is agradient of isopropanol in hexane. Using a small concentration of thepolar solvent in the non-polar solvent (preferably about 0.5%isopropanol in hexane) known tocotrienols and tocopherols may be eluted,while the novel tocotrienols and tocotrienol-like compounds of thisinvention are retained on the amine column. Then, by increasing theconcentration of the more polar solvent (preferably to about 3%isopropanol in hexane), the tocotrienols and tocotrienol-like compoundsof this invention may be selectively eluted, while any sterol and waxycontaminants remain bound to the amine column. Finally, thesecontaminants may be eluted using a high polarity solvent system, such as6-10% isopropanol in hexane. Using this procedure, the desirabletocotrienols and tocotrienol-like compounds may be effectivelyseparated.

An alternative technique also increases the recovered yield oftocotrienols and tocotrienol-like compounds from a biological source. Inthis alternative procedure, a biological source, stabilized orunstabilized, is first extracted with methanol. This step removes manyof the unbound contaminants. Then, heat and, optionally, pressure, areapplied to the biological source to release the tocotrienols andtocotrienol-like compounds. Examples of heat sources include, but arenot limited to radiant heat sources (i.e., visible light or radioactivematerials), convectional heat sources, microwaves, radio-frequency,friction or shearing. The preferred heat source is microwave heat.Alternatively, freeze/thaw methods or mechanical grinding might also beemployed before, during or after heat and/or pressure treatment.Alternatively, the tocotrienols and tocotrienol-like compounds may bereleased by the use of caustic agents, for example, acids, such ashydrochloric or sulfuric acid. According to an alternate embodiment,sonication or detergent treatment may be employed prior to, concurrentlywith, or following the heat, pressure or caustic agent treatment.Experiments using the techniques described herein that employ variouscombinations of heat, pressure and reaction conditions will readilyindicate the preferred conditions for a given biological source.

In purified form, one of the preferred compounds of the presentinvention, P₂₅, exhibits greater biological activity as acholesterol-lowering agent than any of the known tocotrienols: includingP₂₁ (known also as "tocotrienol"), α-T₃, β-T₃, γ-T₃ or δ-T₃. We believethat the double bonds on the isoprenoid side chain comprise the activeportion of these compounds because their presence decreases the effectof London forces between the lipid side chain in biological membranes.We also believe that the number and the position of the alkylsubstituents on the aromatic ring system and the isoprenoid side chainmake a difference in the biological activity--the fewer the number ofalkyl substituents, the greater the activity. We believe that reducingthe steric hindrance caused by the methyl groups allows P₂₅ to penetratemore deeply into the membrane. Accordingly, the membrane may become morehighly organized and thus, less permeable. Under this hypothesis, thecompounds of this invention which substitute hydrogen for methyl at the2-position of the chromanol ring demonstrate increased biologicalactivity. However, alterations at that and other positions are alsoenvisioned, as several different mechanisms which result in increasedbiological activity may be involved.

Because of their ability to lower total serum cholesterol and lowdensity lipoprotein-cholesterol and increase the HDL-/LDL-cholesterolratio, the novel tocotrienols and tocotrienol-like compounds of thisinvention may be used in the prevention and treatment of diseasesassociated with high levels of cholesterol.

Advantageously, the compounds of this invention do not substantiallyalter the serum levels of other blood components that contribute to thebiodegradation of cholesterol. For example, the compounds of thisinvention do not substantially reduce the activity of cholesterol7α-hydroxylase--the enzyme that is responsible for degradation ofcholesterol into bile acids.

Examples of diseases associated with high levels of cholesterol that maybe treated by the compounds of this invention include, but are notlimited to, atherosclerosis, thrombosis, coronary artery disease andother forms of cardiovascular disease. In addition, the ability of thecompounds of this invention to lower serum glucose levels may increasethe insulin production in Type 2 diabetics.

The compounds of this invention may also be used to alter the serum orplasma levels of several other blood constituents. For example, thesecompounds lower the plasma levels of thromboxane A₂ and platelet factor4. In addition, they may serve passively as simple antioxidants oractively by decreasing the release of superoxides by neutrophils andother cytotoxins or cytokines, mast cells, macrophages, endothelialtissue and other immunoregulatory tissues. Antioxidation is accomplishedin at least two ways. First, by reducing arachidonic acid metabolites,the neutrophils reduce the levels of superoxide production. Second,these compounds scavenge radicals which are already present.Accordingly, they exert a protective effect on the endothelium,lipoproteins, smooth muscle cells and platelets. In addition, thesecompounds may also serve as antioxidants to prolong the shelf lives ofproducts prone to oxidation, such as food products. Advantageously, thetocotrienols and tocotrienol-like compounds of this invention may alsoprevent oxidative degradation of food products which results in theformation of carcinogenic compounds in such food products.

The thromboxanes (whose plasma levels are decreased using the compoundsof this invention) also induce platelet aggregation andvasoconstriction. Therefore, the tocotrienols and tocotrienol-likecompounds of this invention may be used to reduce blood clotting in awide variety of applications. For example, these compounds may be usedto treat or prevent diseases, such as thrombotic diseases,cardiovascular diseases, hypertension, pulmonary diseases and renaldiseases. Specifically, the compounds of this invention may be used toprevent or reverse blood clots and lesions which may cause diseases suchas myocardial infarction, stroke, pulmonary embolism, deep veinthrombosis, peripheral arterial occlusion and other blood systemthromboses.

Tocotrienols and tocotrienol-like compounds are also capable of actingas antiatherogenic agents by inhibiting or reversing the oxidation ofLDL and by protecting vascular tissue in general from oxidative damages.The oxidated form of LDL ("OX-LDL") is a major component in theformation of atheroma. Such formation commonly results in a narrowing ofthe arteries by atherosis plaques. While not wishing to be bound bytheory, we believe that by reducing serum LDL levels, tocotrienols andtocotrienol-like compounds enhance the rate of metabolic LDL turnoverand therefore, decrease the exposure of LDL to oxidative agents.

Tocotrienols and tocotrienol-like compounds also decrease theconcentration of lipoprotein (a) in the blood. It has been wellestablished that elevated concentrations of Lp(a) are correlated withearly onset and progression of atherosclerosis, premature myocardialischemia and rheumatoid arthritis. In fact, Lp(a) concentration is amore accurate indicator of coronary heart disease than LDLconcentration. Lowering Lp(a) concentrations in individuals having highLp(a) levels (above about 20 mg/dl) drastically reduces the probabilityof atherosclerosis and coronary heart disease. Significantly, Lp(a)levels, unlike LDL concentration, are not affected by low-fat diets orcommercial available hypocholesterolemic agents.

Tocotrienols and tocotrienol-like compounds also exhibit diureticactivity--they are antagonistic to vasopressin and angiotensin II.Accordingly, these compounds are useful in the treatment and management,for example, of hypertension.

According to another embodiment of this invention, tocotrienol ortocotrienol-like compounds may be administered pre-operatively to apatient in order to prevent septic shock. Advantageously, tocotrienolsand tocotrienol-like compounds may be used in the treatment ofextracorporeal blood. As used herein, the term "extracorporeal blood"includes blood removed in line from a patent subjected to extracorporealtreatment, and returned to the patient in processes such as dialysis, orblood filtration or bypass timing surgery. And the term includes bloodproducts which are stored extracorporeally for eventual administrationto a patient. Such products include whole blood, platelet concentratesand any other blood fraction in which inhibition of platelet aggregationis desired.

According to another embodiment of this invention, tocotrienols andtocotrienol-like compounds may be formulated in compositions and methodsfor coating the surface of invasive devices, to lower the risk ofplatelet aggregation--for example, the surfaces of devices such as,vascular grafts, stents, catheters and artificial valves. Such devicesmay be coated with the tocotrienols and tocotrienol-like compounds usingconventional methodologies including physical adsorption and chemicalcross-linking.

Furthermore, we have found that the tocotrienols and tocotrienol-likecompounds of this invention, the known tocotrienols, and mixturesthereof also reduce the levels of tumor necrosis factor in response tolipopolysaccharide stimulation, lower arachidonic acid in the tissuesand reduce oxygen metabolites in the blood of animals and humans. Theseresults point to an overall reduction in prostaglamdins andleukotrienes, both of which are synthesized from arachidonic acid, and apossible reduction in interleukin-1. Accordingly, the compounds of thisinvention may be employed for a variety of uses. For example, they maybe used to prevent endothelial injury, such as ischemic and reperfusedmyocardium and ulcers. In addition, the inhibition of tumor necrosisfactor biosynthesis would also be accompanied by a decrease ininflammation--i.e., through inhibiting the respiratory bursts ofneutrophils or through free radical scavenging. Therefore, the compoundsof this invention and the known tocotrienols are also useful asantiinflammatory agents for the prevention and treatment of a widevariety of diseases and conditions involving minor, acute and chronicinflammation. These include, but are not limited to, fever, rheumatoiddiseases, pain, functio laesa, hypertension and edema.

In addition to their role in inflammatory response, prostaglandins havealso been shown to inhibit glucose-induced insulin release, increaseglucose concentration and stimulate glucagon secretion. Consequently,use of the compounds of this invention typically leads to an increasedinsulin to glucagon ratio. Therefore, the novel tocotrienols andtocotrienol-like compounds of this invention, the known tocotrienols,and mixtures thereof, may be used to improve glucose intolerance indiabetes mellitus. They may also be used to restore acuteglucose-induced insulin response in non-insulin-dependent diabetesmellitus.

In addition to the above-stated uses, the tocotrienols andtocotrienol-like compounds of this invention, the known tocotrienols,and mixtures thereof, may also be used to enhance the immune response inanimals and humans. These compounds typically reduce the amount of fattyacids in biological tissues. Since fatty acid levels effect the immunesystem, the compounds of this invention may serve as immunoregulators.They may, for example, be used to increase antibody titers to foreignproteins.

In addition, the reductions in fatty acid, cholesterol, triglyceride andglucose levels effected by the compounds of this invention are obtainedwithout attendant substantial weight loss. The result is an increasedfeed to protein conversion ratio. Therefore, the novel tocotrienols andtocotrienol-like compounds of this invention, the known tocotrienols,and mixtures thereof, are useful in increasing feed conversionefficiency.

Hypercholesterolemic diseases and conditions that may be treated usingthe compositions and mixtures described herein include, but are notlimited to, arteriosclerosis, atherosclerosis, xanthomatosis,hyperlipoproteinemias, and familial hypercholesterolemia.

Thrombotic diseases and conditions that may be treated using suchcompositions include, but are not limited to, pulmonary disease, ingeneral (such as reduced specific conductance, reduced dynamiccompliance and constriction (contraction of smooth muscle), excesspulmonary fluids (such as pulmonary lymph, foam, or bronchoalveolarlavage), adult respiratory distress syndrome, astatis and rhiniticdisease (such as pulmonary and systemic hypertension, pulmonary edema,fluid accumulation (neutrophil infiltration) and pulmonary vascularpermeability), pulmonary vasoconstriction (associated, for example, withendotoxemia, gram-negative organisms, anaphylaxis, hemorrhagic shock orallergy to ragweed), cardiac ischemia, microembolic and/or frankocclusion, reocclusion following transluminal angioplasty, myocardialinfarction, cardiopulmonary bypass associated dysfunction,vasoconstriction (pulmonary and peripheral), organ dysfunction, plateletconsumption and/or activation (and subsequent decreased function,aggregation and decreased numbers), mitral valve pathology associatedwith acute perioperative pulmonary hypertension, chronic obstructivearterial disease caused by arteriosclerosis, Maurice Raynaud'ssyndrome--vasoconstriction, renal artery stenosis, myocardialinfarction, stroke, pulmonary embolism, deep vein thrombosis, peripheralarterial occlusion and other blood system thromboses.

Antioxidizing uses include, but are not limited to, the treatment andprevention of endothelial injury, such as ischemic and reperfusedmyocardium. Because of their antioxidizing activity, the tocotrienolsand tocotrienol-like compounds of this invention may also be used intreating and preventing cancerous conditions (by, for example,preventing cancer-causing mutations in the genetic material of an animalor a human).

Antiatherogenic diseases and conditions that may be treated using suchcompositions include, but are not limited to, arteriosclerosis,atherosclerosis, mydocardial infarction, ischemia (i.e., myocardialischemica, brain ischemia and renal ischemia) and strokes.

Inflammatory diseases and conditions that may be treated using suchcompositions include, but are not limited to, essential hypertension,hypertension of congestive heart failure, renal dysfunction caused byreduced myocardia output, endotoxemia, chronic liver disease orhypertension, pulmonary inflammation in asthma, bronchitic, pneumonia oracute lung injury; rheumatic diseases (such as rheumatoid arthritis andsystemic lupus crythematosus), inflammatory bowel disease (such asulcerative colitis), irritable bowel disease (such as villous adenoma),gastrointestinal disorders caused by excess acids, pepsin or bile salts,Zollinger-Ellison syndrome, skin diseases or trauma (such as burns oracid or caustic injury), gout, Bartter's syndrome, fever, rheumatoiddiseases, pain, functio laesa, hypertension and edema.

Immunoregulatory diseases and diseases that may be treated using thecompositions of this invention include, but are not limited to, chronicfatigue syndrome, graft rejections, autoimmune diseases, such as AIDS,and other viral diseases that weaken the immune system.

The compounds and mixtures described herein are useful in pharmaceuticalcompositions, foodstuffs and dietary supplements. Advantageously, theseproducts are hypocholesterolemic, antithrombotic, antioxidizing,antiatherogenic, antiinflammatory and immunoregulatory agents.

Pharmaceutical compositions may take the form of tablets, capsules,emulsions, suspensions and powders for oral administration, sterilesolutions or emulsions for parenteral administration, sterile solutionsfor intravenous administration and gels, lotions and cremes for topicalapplication. The pharmaceutical compositions may be administered tohumans and animals in a safe and pharmaceutically effective amount toelicit any of the desired results indicated for the compounds andmixtures described herein.

This invention also relates to prodrug forms of tocotrienol andtocotrienol-like compounds which, upon administration to a patient,undergo biotransformation into active form.

The pharmaceutical compositions of this invention typically comprise apharmaceutically effective amount of a tocotrienol or tocotrienol-likecompound of this invention, or a mixture thereof, and a pharmaceuticallyacceptable carrier. Such carriers may be solid or liquid, such as, forexample, cornstarch, lactose, sucrose, olive oil or sesame oil. If asolid carrier is used, the dosage forms may be tablets, capsules orlozenges. Liquid dosage forms include soft gelatin capsules, syrup orliquid suspension.

Therapeutic and prophylactic methods of this invention comprise the stepof treating patients in a pharmaceutically acceptable manner with thecompositions and mixtures described herein. As used in this application,the term "pharmaceutically effective amount" or "cholesterol-loweringamount" refers to an amount effective to lower blood levels ofLDL-cholesterol and total serum cholesterol, while increasing the ratioof HDL-cholesterol to LDL-cholesterol in the blood. Alternatively, theterm "pharmaceutically effective amount" refers to an amount effectiveto decrease blood levels of LDL-cholesterol and total serum cholesterolassociated with hypercholesterolemia, an amount effective forlipogenesis, an amount effective to inhibit platelet aggregation, anamount effective to decrease the release of superoxides by humanperipheral blood neutrophils, an amount effective to reduce the level oftumor necrosis factor or interleukin-1, an amount effective to reducethe level of arachadonic acid, an amount effective to increase antibodytiters in the blood, an amount effective for antithrombotic uses, anamount effective to treat, prevent or delay the onset of any one of thefollowing diseases or conditions including inflammatory diseases,immunoregulatory diseases, fever, edema, diabetes mellitus, cancer,signs of aging, pain, septic shock, chronic fatigue syndrome and functiolaesa; or an amount effective to decrease the concentration of Lp(a) inthe blood or to increase feed conversion efficiency.

The pharmaceutical compositions of this invention may be employed in aconventional manner for the treatment and prevention of any of theaforementioned diseases and conditions. Such methods of treatment andprophylaxis and their dosage levels and requirements are well-recognizedin the art and may be chosen by those of ordinary skill in the art fromthe available methods and techniques. Dosage ranges may be from about 1to about 1000 mg/day. However, lower or higher dosages may be employed.Specific dosage and treatment regimens will depend upon factors such asthe patient's health status, the severity and course of the patient'sdisease or disposition thereto and the judgment of the treatingphysician.

Tocotrienols and tocotrienol-like compounds and mixtures thereof mayalso be used in combination with conventional therapeutics used in thetreatment or prophylaxis of any of the aforementioned diseases. Suchcombination therapies advantageously utilize lower dosages of thoseconventional therapeutics, thus avoiding possible toxicity incurred whenthose agents are used as monotherapies. For example, tocotrienols ortocotrienol-like compounds may be used in combination with bile acidsequestrants, such as Cholestyramine and Colestipol; fibric acidderivatives, such as, Clofibrate, Gamfibrozil, Bezafibrate, Fenofibrate,and Ciprofibrate; HMGR inhibitors, such as Lovastatin, Mevastatin,Pravastatin, Simvastatin and SRI-62320; Probucol; Nicotinic Acid; itsderivatives and conjugates, such as, 6-OH-Nicotinic Acid, NicotinariaAcid, Nicotinamide, Nicotinamide-N-oxide, 6-OH-Nictinamide, NAD,N-Methyl-2-pyridine-8-carboxamide, N-Methyl-Nicotinamide,N-Ribosyl-2-Pyridone-S-Carboxide,N-Methyl-4-pyridone-5-carboxamide,Bradilian, Niceritrol, Sorbinicate and Hexanicit; Neomycin andd-Thyroxine.

In foodstuffs, tocotrienols and tocotrienol-like compounds, and mixturesthereof, may be used with any biologically acceptable carrier to providesafe and effective means of lowering blood levels of LDL-cholesterol andtotal serum cholesterol, while increasing the ratio of HDL-cholesterolto LDL-cholesterol in the blood. In addition, the foodstuffs may be usedto inhibit platelet aggregation, to decrease the release of superoxidesby human peripheral blood neutrophils, to reduce the levels of tumornecrosis factor and interleukin-1, to increase antibody titers in theblood, or to treat, prevent or delay the onset of one or more of thefollowing diseases or conditions: immunoregulatory disease,inflammation, fever, edema, diabetes mellitus, cancer, signs of aging,pain, rheumatoid diseases, septic shock, chronic fatigue syndrome andfunctio laesa. Alternatively, they may be used to decrease Lp(a)concentrations in the blood or to increase feed conversion efficiency.

Foodstuffs containing the tocotrienols and tocotrienol-like compoundsaccording to this invention, and mixtures thereof, may be combined withany other foodstuff. For example, oils containing the compounds of thisinvention may be used as cooking oil, frying oil, or salad oil and maybe used in any oil-based food, such as margarine, mayonnaise or peanutbutter. Grain flour fortified with the compounds of this invention maybe used in foodstuffs, such as baked goods, cereals, pastas and soups.Oils containing tocotrienols and tocotrienol-like compounds can beemulsified and used in a variety of water-based foodstuffs, such asdrinks, including drink mixes. Advantageously, such foodstuffs may beincluded in low fat, low cholesterol or otherwise restricted dietaryregimens.

The pharmaceutical compositions and foodstuffs of this invention may beadministered to humans and animals such as, for example, livestock andpoultry. Once an animal has ingested or otherwise been administered atocotrienol or tocotrienol-like compound, or a mixture thereof, itadvantageously retains the hypercholesterolemic, antithrombotic,antioxidizing, antiinflammatory, antiatherogenic, immunoregulatory andother advantageous biological activities of the administered compounds.Therefore, such an animal, or any product derived therefrom, such as,for example, milk, may be consumed by a human or another animal toderive the benefits of the tocotrienols and tocotrienol-like compounds.For example, a chicken which ingests a grain or meal fortified with thecompounds of this invention may later be eaten by a human to derive thecholesterol-reducing benefits.

In addition, the administration of tocotrienols or tocotrienol-likecompounds to animals results in an increase in feed conversionefficiency. In higher fat content animals, such as cattle, swine, sheepand lamb, tocotrienols or tocotrienol-like compounds advantageously leadto faster growth, lower cholesterol content, and higher percentage leanmeat. When administered to fowl, tocotrienols and tocotrienol-likecompounds result in production of eggs characterized by reducedcholesterol content of the yolk and higher protein content of the eggwhite.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any way.

In these examples, mass spectra data was acquired using Model MS-902manufactured by and obtainable from Associate Electrical Industries,Ltd. (Department of Chemistry, University of Wisconsin, Madison).Samples were introduced directly into the ion source on a glass probe at100° C. The potential of the ionizing electrical beam was 70 e.v. NMR inCDCl₃ were obtained using NMR-500 by BiuKr, FDR at room temperature. IRwas done as thin film using a Perkin Elmer instrument. Centrifugationwas done in an Eppendorf Centrifuge (model 1240). Microwaving was donein a Whirlpool household oven (Model No. MT6901XW-O operating at 800watts. HPLC was done using a Waters pump 6000A or a Gilson pump 307, aShimadzu fluorescence detector RF-535 and integrator CR3A, a Gilson'sautoinjector, Perkin Elmer 50A UV detector or a Shimadzu SPD-10 AVUV-VIS spectrum photometric detector and a Kipp and Zonen BD41 recorder.Unless otherwise indicated, the following HPLC conditions were employed:20 μl injections, Porasil column (Waters column, 10μ, 4 mm×30 cm) usingan isocratic system of hexane and isopropanol (99.75%:0.25%, v/v) at aflow rate of 1.3 ml/min. Detection was done at an exitation wavelengthof 295 nm and an emission wavelength of 330 nm (fluorescence detector)and UV absorption at 295 nm.

All assays conducted on chicken or swine serum were done following theprotocols described in A. A. Qureshi et al., "Lowering Of SerumCholesterol In Hypercholesterolemic Humans By Tocotrienols" (Palmvitee),Am. J. Clin. Nutr., 53, pp. 1021S-26S (1991). All enzymatic assays weredone following the protocols described in A. A. Qureshi et al., "Effectsof Cereals and Culture Filtrate of Trichoderma viride on LipidMetabolism of Swine", Lipids, 17, pp. 924 (1982). Plasma insulin,glucagon and TNF levels were measured using radioimmunoassay kits fromVentrex Laboratories, Inc. (Portland, Me.), ICN Biomedicals, Inc. (CostaMesa, Calif.) and Genzyme Corp. (Cambridge, Mass.), respectively.

Example 1

The following protocol may be employed for carrying out thestabilization of a biological source, using rice bran as the sample.

Rough rice (paddy rice) from a farm is dried in a commercial-typecontinuous flow, non-mixing, heated air dryer. Drying is carried out tolower the moisture content of the rice from a level of between about 18and 22 percent to a level between about 10 and 13 percent. The driedrice is then cleaned by removing dust, stones, seeds and sticks byaspiration in a commercial rice cleaning machine, followed by gravityseparation in a stoner and particle size separation in a disk grader anda drum separator. The husks are then removed using a rubber rollersheller. Paddy (husks or hulls) are removed using a paddy separator. Theraw bran is then removed in a friction mill to yield polished rice. Theraw bran is then pneumatically conveyed either to the extruder or tostorage until stabilization.

To carry out stabilization, the raw bran is pneumatically conveyed to afilter/sifter to remove residual broken rice. After sifting, the rawbran is pneumatically conveyed to a mixing/tempering hopper tank. Theraw bran is conveyed from the discharge of the mixing tank to theextruder inlet valve of a clamped barrel single screw extruder by ametered screw conveyer feeder. The operating conditions of the extruderare maintained during stabilization in the following range:

flow rate: 900-2000 lbs/hr

pressure: 800-2000 PSI

temperature: 135°-210° C.

time: 5-90 seconds.

The dry heat stabilized raw bran is then fed directly into the feedhopper of an expander cooker. Alternatively, the feed to the cooker maybe raw bran that has been cooled with dry ice. Within the extruder, thebran is conveyed by a discontinuous worm shaft toward the dischargeplate at a rate of around 341 lbs/hr. Water and steam are added throughinjection ports in the barrel of the extruder at a rate of around 38lbs/hr to completely mix the material and to raise the moisture level.The ambient temperature is about 30° C. Flow of the material iscontrolled by a discharge die plate at a rate of about 341 lbs/hr. Thefeed material moisture level is maintained at about 11.4% and thetemperature is held between about 90° and 135° C. for between about 15and 90 seconds. The moisture level in the collet produced is about 14%at a discharge temperature of 125° C.

As the bran is extruded through the die plate, the sudden decrease inpressure causes the liquid water to vaporize. During cooking, enzymesare denatured and some constituents of the bran are gelatinized into afluid paste which binds the particles together. A compact pellet isformed. Vaporization of water causes breakage within the cells ideallysuited for solvent migration percolation. The introduction of steam andwater during the process raises the moisture content of the bran toabout 22-25 percent. The extruder discharge is then sent downstream at arate of around 341 lbs/hr. to a dryer/cooler. Moisture flow to theextruder is maintained at about 96 lbs/hr and the temperature is kept inthe range of 82° C. to 130° C. The discharge from the dryer/cooler ismaintained at a rate of about 341 lbs/hr and at a moisture level ofabout 8%. Stabilized rice bran is the result. These conditions alsoallow for storage of the stabilized bran.

The stabilized bran is immersed in hexane in a ratio by weight of abouttwo to one. Typically, about 10-100 g of material can be extracted usingthis protocol. The hexane is generally heated to about 60° C. using asteam table incorporated into an explosion proof vented hood, but othersolvents and other temperatures may also be employed. The hexane/oilmiscella is removed from the bran by filtration. About 5-6 washings arenecessary to bring the oil content of the bran to less than one percent.The defatted bran and the hexane/oil miscella are both desolventizedunder gentle heating with steam.

If 100-500 lbs. of stabilized bran is to be extracted, it is morepractical to use the following protocol. The stabilized bran is fed intoa counter-current extractor at a flow rate of about 111 lbs/hour. Freshhexane is introduced at a rate of around 312 lbs/hr. The fresh solvent(hexane) temperature is maintained at about 50° C., while the extractortemperature is maintained at around 52° C. Residence time in theextractor is typically around 45 minutes. The product is a defatted branwith an oil content of less than one percent. The hexane/oil miscellaexiting the discharge of the extractor is filtered through a plate andframe filter press. The filtered miscella is then pumped to a steamheated still where the hexane is evaporated and collected by a condenserfor reuse.

Following extraction and desolventization, the crude rice bran oil istypically degummed, dewaxed, bleached and physically refined using steamdistillation. Degumming is carried out by a two stage addition underagitation of 2% water by weight and then 0.15% phosphoric acid (85%reagent grade) by weight. The temperature is held at about 82° C. to 88°C. for 10 minutes and the sludge containing the gums is then removed viaultrafugation. (See, e.g., U.S. Pat. No. 4,049,686). The degummed branis cooled to about 5° C. to 8° C. and held for 24 hours. The dewaxedoils form a layer above the waxes which can be decanted using a vacuumpump. Bleaching is carried out according to the official American OilChemist's Society method 6c 8a-52. Physical refining is carried out in aglass deodorizer at about 250° C. and around 3 mm Hg for about 2 hours.

The following specific protocols are referred to in the subsequentexamples. Stabilization protocols follow the general method set forthabove, with the precise conditions defined as follows:

    ______________________________________                                        Protocol I - Dry Heat Stabilization                                           Extruder: Wenger Model X-25                                                   Standard Screw/Barrel Setup:                                                  Barrel # Standard Port                                                                              Screw #  Standard Port                                  ______________________________________                                        5        28714-9      5        28320-1                                        4        28318-1      4         8326-9                                        3        28372-9      3        28326-1                                        2        28318-1      2        28326-5                                        1        28350-1      1        28387-9                                        Standard Die Setup:                                                           Die/Spacer    Measurement                                                                              Standard Port                                        ______________________________________                                        Spacer        0.375      28340-11                                             Back Plate    0.625      28361-51                                             Intermediate  0.218      28316-723                                            Plate                                                                         Front Plate   0.235      28389-507                                            Operating Conditions:                                                         Feed Rate:          1000 lbs/hr                                               Temperature:        170° C. at exit die                                Pressure:           975-1025 psi                                              Moisture Feed:      12%                                                       Moisture Discharge: 9.6%                                                      Residence Time:     15 seconds                                                Run Duration:       8 hours                                                   Sample Size:        50 lbs.                                                   Protocol II-Dry Heat Followed by Wet Heat Stabilization                       Dry Heat Stage: Protocol I                                                    Wet Heat Stage:                                                                Extruder: Anderson 4 inch                                                     Screw/Barrel Configuration: Standard Cut Flight                               Die Setup:                                                                     Diameter: 0.1875 inches                                                       Land: 0.75 inches                                                           Overating Conditions:                                                         Feed Rate:       378 lbs/hr                                                   Shaft Speed:     279 rpm                                                      Steam injection: 36 lbs/hr (32 psi at #8 hole)                                Mechanical Pressure:                                                                           750 psi (ast.)                                               Moisture Feed:   11.4%                                                        Discharge Moisture:                                                                            15%                                                          Discharge Rate:  450 lbs/hr                                                   Discharge Temp.: 121° C.                                               ______________________________________                                    

Protocol III-Drying/Cooling Procedure

The wet heat stabilized product of protocol II (15% moisture) wasdischarged onto aluminum trays and placed in a tray oven at 101.1° C.until the moisture content was 8-10% (approximately 1.5 hours). Thetrays were then placed on tray racks and allowed to cool at ambienttemperature (approximately 20° C.).

    ______________________________________                                        Protocol IV-Oil Extraction (Laboratory Method)                                ______________________________________                                        Oil to hexane ratio:   1:4                                                    # of washings:         3                                                      Extraction temperature:                                                                              40° C.                                          ______________________________________                                    

The hexane was removed from the extract by mild heating (40° C.) under amild vacuum.

    ______________________________________                                        Protocol V-Oil Extraction (Pilot Plant Method)                                ______________________________________                                        Oil to hexane ratio:                                                                              1:4                                                       # of washings:      6                                                         Extraction temperature:                                                                           60° C.                                             Amount Extracted:   20 lbs                                                    Yield:              16 lbs defatted bran                                                          4 lbs crude oil                                           ______________________________________                                    

The hexane was removed from the extract by heating to 115.5° C.

    ______________________________________                                        Protocol VI-Oil Extraction (Cold Extraction)                                  ______________________________________                                        Oil to hexane ratio:                                                                             1:4                                                        # of washings:     6                                                          Extraction temperature:                                                                          20° C. (ambient)                                    Amount Extracted:  20 lbs                                                     Yield:             16.4 lbs defatted bran                                                        3.6 lbs crude oil                                          ______________________________________                                    

The hexane was removed from the extract by heating to 115.5° C.

Protocol VII-Dewaxing

20 lbs of crude oil were refrigerated for 24 hours at 4° F. (-15.6° C.).The supernatant, which contained the dewaxed oil, was decanted from thesolidified waxes. The waxes were then centrifuged to remove entrainedoil yielding waxes (0.59 lbs) and dewaxed oil (19.407 lbs).

Example 2 - Purification of P₂₅

The rice bran was stabilized according to protocol I. 1.0 g of thestabilized rice bran was ground into a fine powder and extracted with 8ml of methanol in a disposable screw capped tube using a shaker for.sup.˜ 30 minutes. The suspension was then centrifuged at 2000 rpm for10 min. to recover the methanol layer, which was evaporated under vacuumat 40° C. The remaining dried residue was then extracted with 4 mlhexane by shaking on a shaker for 3 min. followed by centrifugation at2000 rpm for 5 min. The resulting supernatant was transferred into aninjecting vial, which was capped and centrifuged again for 2 min.Evaporation yielded the TRF.

Ten mg of the TRF, which contained novel tocotrienol P₂₅, was dissolvedin 0.5 ml of hexane and bound to a Bond Elute Amine column (1.0 ml) thatwas first equilibrated with 2 ml hexane. The column was then washed with1 ml of hexane, followed by 1 ml of 3% isopropanol in hexane. This stepremoved most of the known Tocol products. Then 1.0 ml of 5% isopropanolin hexane was run through the column. The recovered solution wasevaporated, yielding 1.85 mg of P₂₅ and 0.44 g of tocotrienol (P₂₁).Finally, 1.0 ml of 6% isopropanol in hexane followed by 1.0 ml 10%isopropanol in hexane was run through the column. The resultantfractions were analyzed by HPLC. No trace of the P₂₀ impurity wasvisible on the HPLC trace.

Example 3

Amine column purified compounds from Example 2 were further purified byHPLC to separate tocotrienol (P₂₁) and P₂₅. The peak fractions fromseveral runs were collected and analyzed by HPLC. The purified sampleseach showed a single symmetrical peak as observed by UV andfluorescence. The following analytical data was obtained for tocotrienol(P₂₁) and P₂₅ :

Tocotrienol P₂₁ :

MS(EI): m/e 382⁺ (molecular ion)

163⁺

123⁺

NMR (CDCl₃):

δ 1.26 (s, H), 1.50-1.84 (m, 4H), 1.58 (s, 9H), 1.66 (s, 3H), 1.90-2.15(m, 10H), 2.69 (t, J=6.7 Hz, 2H), 4.23 (s, 1H), 5.07 (m, 3H), 6.51-6.57(m, 2H), 6.63 (d, J=8.5 Hz, 1H).

IR (film):

(cm⁻¹) 3400, 2990, 2950, 2876, 1500, 1458, 1240, 750.

The MS data correspond to a molecular formula of C₂₆ H₃₈ O₂. The patternis characteristic of the fragmentation observed with α-, β-, andγ-tocotrienols as reported by Rao et al., "Identification and Estimationof Tocopherols and Tocotrienols in Vegetable Oils Using GasChromatography-Mass Spectrometry", J. Agr. Food Chem., 20(2), pp.240-245(1972) . The peak at m/e 163⁺ indicates loss of the side chain (C₁₆H₂₇)⁺ giving rise to the ion C₁₀ H₁₁ O₂ (163.0759). The peak m/e 123⁺originates from the cleavage of the side chain by the breakdown of thechroman structure with hydrogen rearrangement and loss of a methylacetylene fragment (CH₃ --C═CH₂).

The NMR spectra is identical to that of T₃, except that the methyl groupon the benzene ring at δ 2.2 normally present in T₃ is missing in thespectrum of P₂₁.

The IR indicates the presence of a phenolic OH group at 3700 cm⁻¹, abenzene ring at 1650 cm⁻¹ and a chroman ring at 1680 cm⁻¹.

Based on these data, the structure of P₂₁ was identified asdesmethyl-tocotrienol(3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3' (E),7'(E),11'-trienyl)-2H-1-benzopyran-6-ol): ##STR6## P₂₅ :

MS(EI):

m/e 368⁺ (molecular ion)

175⁺

149⁺

123⁺

NMR (CDCL₃):

δ 1.58 (s, 3H), 1.59 (s,3H), 1.62 (s, 3H), 1.67 (s, 3H), 1.73 (m, 2H),1.90-2.08 (m, 10H), 2.19 (m, 2H), 2.63-2.85 (m, 2H), 3.89 (m, 1H), 4.30(br. s, 1H), 5.07 (m, 2H), 5.14 (t, J=7.9 Hz, 1H), 6.51 (d, J=2.9 Hz,1H), 6.56 (d of d, J=2.9, 8.6 Hz, 1H), 6.66 E(d, J=8.6 Hz, 1H).

IR (film):

(cm⁻¹ ) 3388, 2924, 1494, 1450, 1352, 1280, 1218, 1080.

The MS data correspond to a molecular formula of C₂₅ H₃₆ O₂. The patternis characteristic of the fragmentation observed for γ-tocotrienol,except that the methyl on the chroman ring adjacent to oxygen ismissing.

The NMR spectra is similar to that of desmethyl-T₃.

The IR indicates the presence of a phenolic OH group at 3388 cm⁻¹, abenzene ring at 1494 cm⁻¹ and a chroman ring at 1352 cm⁻¹.

Based on these data, the structure of P₂₅ was identified asdidesmethyl-tocotrienol (3,4-dihydro-2-(4,8,12-trimethyltrideca-3'(E),7' (E),11'-trienyl)-2H-1-benzopyran-6-ol): ##STR7##

Example 4 - Isolation and Purification of P₁₆ and P₁₈

The rice bran was stabilized according to protocol I. 1.0 g of thestabilized rice bran was ground into a fine power and microwaved for 3minutes (in 1 minute intervals) to release any bound T₃ and T₃ -likecompounds. The microwaved rice bran was then extracted with 7 ml ofmethanol in a disposable screw capped tube using a shaker for .sup.˜ 30minutes. The suspension was then centrifuged at 2000 rpm for about 5minutes. The supernatant was removed and the methanol was evaporatedunder vacuum at 40° C. The resulting residue contained a mixture of freeand previously bound T₃ and T3-like compounds.

The residue was dissolved in 250 μl (200 mg) of hexane and applied to a1 ml Bond Elute amine column that was first equilibrated with 2 ml ofhexane. The column was then washed with 1 ml of hexane. Then 0.5 mlaliquots of the following solvents were eluted through the columnsuccessively: 0.5% isopropanol in hexane, 1.0% isopropanol in hexane,1.5% isopropanol in hexane, 2.0% isopropanol in hexane, 2.5% isopropanolin hexane and 3% isopropanol in hexane. Finally, the column was washedwith 0.5 ml of 10% isopropanol in hexane. The resultant fractions wereanalyzed and the fractions containing P₁₆ and P₁₈ were combined (onlythe fractions collected using 1.5%-3% isopropanol in hexane containedP₁₆ and P₁₈ in significant amounts).

In order to separate P₁₆ and P₁₈, HPLC conditions identical to thosedescribed in this application were used, except that the concentrationof isopropanol was increased from 0.25% to 0.5% in hexane. Under thoseconditions, P₁₆ eluted at 13 minutes and P₁₈ eluted at 15 minutes. Thepeak fractions were collected and analyzed by HPLC. These purifiedsamples each showed a single symmetrical peak as observed by UV andfluorescence. The following analytical data was obtained for P₁₆ and P₁₈:

P₁₆ :

MS(EI): m/e 380+ (molecular ion)

161+

molecular formula: C₂₇ H₄₀ O

molecular weight (actual): 380.3084

(calculated): 380.3079

The fragmentation pattern was similar to that of P₂₁ and P₂₅. The peakat m/e 161+ indicates loss of the C₁₆ H₂₇ side chain giving rise to C₁₁H₁₃ O. Based on these data, the following structure for P₁₆ wasidentified: ##STR8## P₁₈ :

MS (EI): m/e 424+ (molecular ion)

205+

molecular formula: C₂₈ H₄₀ O₃

molecular weight (actual): 424.3010

(calculated: 424.2977

The fragmentation pattern was again Similar to that of P₂₁ and P₂₅.Based upon these data, the following structure for P₁₈ was identified:##STR9##

Example 5

The effects of different rice brans on the serum levels of totalcholesterol, HDL-cholesterol, LDL-cholesterol apolipoprotein (a)₁ andapolipoprotein B was determined in chickens.

The following protocols were performed to yield the samples displayed inTable I:

entry 5: protocol I

entry 6: protocol I, followed by protocol IV.

All other samples were obtained from commercially available sources.

Chickens were chosen as our initial model because they synthesizecholesterol in the liver using ε mechanism similar to that of humans(unlike rats and mice that synthesize cholesterol in the intestines).Each group of chickens (6-week old female white leghorn) wasadministered either a chick mash control diet or a control dietcontaining a 20% equivalent of a test rice bran. The amount of feedconsumed by all groups was comparable to the control group and thefeeding period was 4 weeks. The birds were fasted for a period of 14hours prior to sacrifice (at 0800 hours).

The chicken mash diet contained the following ingredients:

    ______________________________________                                        Ingredients       Weight (g)                                                  ______________________________________                                        Corn (8.8% protein)                                                                             615.0                                                       Soybean Meal      335.0                                                       Corn Oil          10.0                                                        Calcium Carbonate 10.0                                                        Dicalcium Phosphate                                                                             20.0                                                        Iodized Salt      5.0                                                         Mineral Mixture.sup.a                                                                           2.5                                                         Vitamin Mixture.sup.b                                                                           2.5                                                         ______________________________________                                         .sup.1 Mineral mixture contained per kg feed: zinc sulfate.H.sub.2 O, 110     mg; manganese sulfate 5H.sub.2 O, 70 mg; ferric citrate.H.sub.2 O, 500.0      mg; copper sulfate.5H.sub.2 O, 16.0 mg; sodium selenite, 0.2 mg;              DLmethionine, 2.5 g; choline chloride (50%), 1.5 g; ethoxyquin                (1,2dihydro-6-ethoxy-2,2,4-trimethylquinoline), 125 mg; and thiamineHCl,      1.8 mg.                                                                       .sup.b Vitamin mixture contained per kg feed: vitamin A, 1,500 units;         vitamin D.sub.3, 400 units; vitamin E, 10 units, riboflavin, 3.6 mg;          calcium pantothenate, 10.0 mg; niacin, 25.0 mg; pyridoxineHCl, 3.0 mg;        folacin, 0.55 mg; biotin, 0.15 mg; vitamin B.sub.12, 0.01 mg; and vitamin     K.sub.1, 0.55 mg.                                                        

The results are displayed below in Table I. Percentages of increases ordecreases are shown in parentheses.

                                      TABLE 1                                     __________________________________________________________________________                  Concentration in Serum (mg/100/ml)                              Nutritional State.sup.1                                                                     Total Chol.                                                                          HDL-Chol.                                                                            LDL-Chol.                                                                             Apo-A.sub.1                                                                          Apo-B                              __________________________________________________________________________    1)                                                                              Control Group Chick                                                                       165.9 ± 6.9.sup.A                                                                 104.2 ± 4.65.sup.A                                                                47.9 ± 4.64.sup.A                                                                  132.9 ± 3.88.sup.A                                                                31.4 ± 1.68.sup.A                 Mash (CTL)  (100.0).sup.2                                                                        (100.0).sup.2                                                                        (100.0).sup.2                                                                         (100.0).sup.2                                                                        (100.0).sup.2                      2)                                                                              Cellulose (20% Equ.)                                                                      155.4 ± 3.5.sup.B                                                                  96.0 ± 4.99.sup.A                                                                42.6 ± 4.12.sup.B                                                                  126.8 ± 3.81.sup.A                                                                30.4 ± 1.31.sup.A                             (93.7) (92.1) (88.9)  (95.4) (96.8)                             3)                                                                              California Rice Bran                                                                      151.2 ± 3.7.sup.B                                                                 100.4 ± 5.42.sup.A                                                                39.0 ± 4.22.sup.A,B                                                                125.8 ± 4.78.sup.A                                                                28.8 ± 1.54.sup.A                 (20% Equ.)  (91.1) (96.4) (81.4)  (94.7) (91.7)                             4)                                                                              Louisiana Rice Bran                                                                       142.0 ± 4.3.sup.C                                                                 101.7 ± 6.41.sup.A                                                                34.6 ± 3.09.sup.B                                                                  132.0 ± 3.84.sup.A                                                                27.6 ± 1.41.sup.B                 (20% Equ.)  (85.6) (97.6) (72.2)  (99.3) (87.9)                             5)                                                                              Louisiana Rice Bran                                                                       128.5 ± 4.0.sup.D                                                                  98.1 ± 7.25.sup.A                                                                31.4 ± 2.72.sup.B                                                                  133.2 ± 3.32.sup.A                                                                25.7 ± 2.25.sup.B                 Stabilized  (77.5) (94.1) (65.6)  (100.2)                                                                              (81.8)                               (20% Equ.)                                                                  6)                                                                              Louisiana Rice Bran                                                                       148.5 ± 6.1.sup.D                                                                 100.2 ± 4.69.sup.A                                                                42.4 ± 3.01.sup.A                                                                  125.7 ± 5.56.sup.A                                                                28.7 ± 1.31.sup.A                 Defatted (20% Equ.)                                                                       (89.5) (96.2) (88.5)  (94.6) (91.4)                             7)                                                                              Commercial Rice Bran                                                                      150.0 ± 4.6.sup.B                                                                  98.9 ± 6.89.sup.A                                                                40.6 ± 3.88.sup.A                                                                  125.1 ± 5.56.sup.A                                                                29.0 ± 1.26.sup.A                 (20% Equ.)  (90.4) (94.9) (84.8)  (94.1) (92.4)                             __________________________________________________________________________     .sup.1 Feeding period was 4 weeks; time of killing was 0800 hours. The        birds were fasted for 14 hours prior to killing. Data expressed as means      ± SD; n = 6 chickens per group.                                            .sup.2 Percentages of increases or decreases are in parentheses.              .sup.A-D Values not sharing a common superscript letter are different at      < 0.01.                                                                  

The diet which included stabilized rice bran demonstrated superiorcholesterol reducing activity when compared with the other diets. Itadvantageously lowered the LDL-cholesterol level by almost 35%, whilelowering the HDL-cholesterol level by only 5.9%, as compared to thecontrol diet. In addition, it increased the ratio of apolipoprotein (a)₁to apolipoprotein B by 21%. This ratio is used as an indicator forassessment of risk for coronary heart disease (see Naito et al., "TheClinical Significance Of Apolipoprotein Measurements," J. Clin.Immunoassay, 9(1), pp. 11-20 (1986)).

Example 6

We next determined the effects of different rice brans on the hepaticenzymatic activities of HMG-CoA reductase, cholesterol 7α-hydroxylaseand fatty acid synthetase, and the serum levels of triglycerides andglucose in chickens fed with them. The feeding conditions were identicalto those described in Example 5. The samples were prepared as describedin Example 5.

The results are displayed below in Table II. Percentages of increasesand decreases are shown in parentheses.

                                      TABLE II                                    __________________________________________________________________________    Hepatic Enzymatic Activities                                                                HMG-CoA  Cholesterol                                                                            Fatty Acid                                                                             Serum   Serum                                      Reductase                                                                              7α-Hydroxylase                                                                   Synthetase                                                                             Triglycerides                                                                         Glucose                      Nutritional State.sup.1                                                                     (pmoles/mg/min).sup.3                                                                  (nmoles/mg/min).sup.4                                                                  (nmoles/mg/min).sup.5                                                                  (mg/100 ml)                                                                           (mg/100                      __________________________________________________________________________                                                     ml)                          1)                                                                              Control Group Chick                                                                       405.3 ± 15.5.sup.A                                                                  0.808 ± 0.062.sup.A                                                                 69.0 ± 3.40.sup.A                                                                   75.7 ± 3.34.sup.A                                                                  109.8 ± 3.50.sup.A          Mash (CTL)  (100.0).sup.2                                                                          (100.0).sup.2                                                                          (100.0).sup.2                                                                          (100.0).sup.2                                                                         (100.0).sup.2                2)                                                                              Cellulose (20% Equ.)                                                                      417.0 ± 14.6.sup.A                                                                  0.882 ± 0.031.sup.A                                                                 58.7 ± 2.25.sup.B                                                                   69.4 ± 2.93.sup.B                                                                  100.9 ± 1.89.sup.B                      (102.9)  (109.2)  (85.1)   (91.7)  (91.8)                       3)                                                                              California Rice Bran                                                                      340.2 ± 17.6.sup.B                                                                  0.851 ± 0.093.sup.A                                                                 55.1 ± 4.59.sup.B                                                                   65.4 ± 2.84.sup.B,C                                                                 96.1 ± 0.94.sup.A,B        (20% Equ.)  (83.9)   (105.3)  (79.9)   (86.4)  (87.5)                       4)                                                                              Louisiana Rice Bran                                                                       330.5 ± 12.8.sup.B                                                                  0.935 ± 0.044.sup.B                                                                 68.7 ± 3.88.sup.A                                                                   63.2 ± 1.61.sup.C                                                                   93.1 ± 2.20.sup.C          (20% Equ.)  (81.5)   (115.7)  (99.6)   (83.5)  (84.8)                       5)                                                                              Louisiana Rice Bran                                                                       311.7 ± 14.6.sup.B                                                                  0.939 ± 0.039.sup.B                                                                 65.6 ± 5.69.sup.A                                                                   63.9 ± .92.sup.C                                                                    90.4 ± 1.38.sup.C          Stabilized (20%                                                                           (76.9)   (116.2)  (95.1)   (84.5)  (82.3)                         Equ.)                                                                       6)                                                                              Louisiana Rice Bran                                                                       388.7 ± 14.8.sup.C                                                                  0.852 ± 0.028.sup.A                                                                 70.4 ± 2.12.sup.A                                                                   72.3 ± 1.67.sup.A                                                                   99.3 ± 1.63.sup.A          Defatted (20% Equ.)                                                                       (95.9)   (105.4)  (102.0)  (95.6)  (90.4)                       7)                                                                              Commercial Rice Bran                                                                      371.2 ± 15.5.sup.C                                                                  0.850 ± 0.036.sup.A                                                                 64.7 ± 3.66.sup.A                                                                   70.9 ± 1.61.sup.A                                                                  101.2 ± 1.21.sup.A          (20% Equ.)  (91.6)   (105.2)  (93.8)   (93.7)  (92.1)                       __________________________________________________________________________     .sup.1 Feeding period was four weeks; time of killing was 0800 hours. The     birds were fasted for 14 hours prior to killing. Data expressed as means      ± SD; n = 6 chickens per group.                                            .sup.2 Percentages of increases or decreases are in parentheses.              .sup.3 pmoles of mevalonic acid synthesized per minute per mg of              microsomal protein.                                                           .sup.4 nmoles of [.sup.14 ccholesterol into [.sup.14 c]                       7hydroxycholesterol per minute per mg of microsomal protein.                  .sup.5 nmoles of NADPH oxidized per minute per mg of cytosolic protein.       .sup.A-C Values not sharing a common superscript letter are different at      < 0.01.                                                                  

As shown in Table II, the diet which contained stabilized rice branproduced a marked reduction (over 23% as compared to the control diet)in the activity of HMG-CoA reductase, the rate-limiting enzyme ofcholesterol biosynthesis. However, the activity of the enzymeresponsible for the degradation of cholesterol, cholesterol7α-hydroxylase, was not substantially affected. Furthermore, this dietreduced the serum levels of triglycerides (fats) and glucose by 15.5%and 17.7%, respectively.

Example 7

We next compared the effects of rice bran oils extracted from varioussources on serum lipid parameter of chickens fed with diets supplementedwith those oils. Each group of 6 chickens (6-week old female whiteleghorn) was fed the chicken mash diet described in Example 5 for 14days. Following this period, the chickens were fed a diet consisting ofthe chicken mash diet containing a 5% supplement of various oils. Thecontrol diet included a supplement of 5% corn oil. After 4 weeks, thebirds were fasted for 36 hours and then refed for 48 hours prior tosacrifice (at 0800 hours). The amount of feed consumed by all groups wascomparable to the control group. The following protocols were performedto yield the samples displayed in Table III:

entry 2: protocol I, followed by protocol IV

entry 3: protocol III, followed by protocol IV

entry 4: protocol III, followed by protocol IV

entry 5: protocol IV

entry 6: protocol IV

All other samples were obtained from commercially available sources.

The results are displayed below in Table III. Percentages of increasesor decreases are shown in parentheses.

                                      TABLE III                                   __________________________________________________________________________                  SERUM CHOLESTEROL (mg/100/ml)                                   Nutritional State.sup.1                                                                     Total Chol.                                                                          HDL-Chol.                                                                            LDL-Chol.                                                                            Triglycerides                                                                         Glucose                            __________________________________________________________________________    1)                                                                              Chick Diet + 5.0%                                                                         185.1 ± 1.5.sup.A                                                                 110.3 ± 4.95.sup.A                                                                61.9 ± 4.49.sup.A                                                                 90.2 ± 2.17.sup.A                                                                  124.6 ± 2.30.sup.A                Corn Oil (CDC)                                                                            (100.0).sup.2                                                                        (110.0).sup.2                                                                        (100.0).sup.2                                                                        (100.0).sup.2                                                                         (100.0).sup.2                      2)                                                                              Chick Diet + 5.0%                                                                         129.7 ± 4.1.sup.B                                                                  99.8 ± 3.57.sup.A                                                                27.9 ± 2.60.sup.B                                                                 84.5 ± 2.97.sup.B                                                                  117.3 ± 5.92.sup.A,B              Louisisana Rice Bran                                                                      (70.7) (90.5) (45.1) (93.7)  (94.1)                               Oil (LRBO)                                                                  3)                                                                              Chick Diet + 5.0%                                                                         135.3 ± 4.5.sup.B                                                                 102.6 ± 4.93.sup.A                                                                30.0 ± 2.89.sup.B                                                                 81.3 ± 3.94.sup.B,C                                                                112.5 ± 4.65.sup.B                Hot Extracted LRBO                                                                        (73.1) (93.0) (48.5) (90.1)  (90.2)                             4)                                                                              Chick Diet + 5.0%                                                                         121.6 ± 3.5.sup.C                                                                  98.4 ± 4.99.sup.A                                                                26.1 ± 2.89.sup.B                                                                 77.2 ± 4.81.sup.C                                                                  113.2 ± 4.40.sup.B                Cold Extracted LRBO                                                                       (65.7) (89.2) (42.2) (85.6)  (90.9)                             5)                                                                              Chick Diet + 5.0%                                                                         154.5 ± 3.4.sup.D                                                                 108.6 ± 3.25.sup.A                                                                46.7 ± 3.25.sup.C                                                                 86.6 ± 4.64.sup.A,B                                                                116.5 ± 6.21.sup.B                Louisisana Crude Rice                                                                     (83.5) (98.5) (75.4) (96.0)  (93.5)                               Bran Oil                                                                      Unstabilized                                                                6)                                                                              Chick Diet + 5.0%                                                                         163.3 ± 5.3.sup.E                                                                 102.4 ± 4.10.sup.A                                                                55.8 ± 3.84.sup.D                                                                 88.5 ± 4.53.sup.A                                                                  116.2 ± 4.78.sup.B                Commercial Rice Bran                                                                      (88.2) (92.8) (90.1) (98.1)  (93.3)                               Oil                                                                         __________________________________________________________________________     .sup.1 Feeding period was 4 weeks; time of killing was 0800 hours. The        birds were fasted for 14 hours prior to killing. Data expressed as means      ± SD; n = 6 chickens per group.                                            .sup.2 Percentage of increases or decreases are in parentheses.               .sup.A-E Values not sharing a common superscript letter are different at      < 0.01.                                                                  

The diet supplemented with 5% cold extracted Louisiana rice bran oildemonstrated superior cholesterol-lowering ability over the other diets.Impressive decreases in total serum and LDL-cholesterol (34.3% and57.8%, respectively) were recorded, while the level of HDL-cholesteroldecreased only slightly (10.8%). Decreases in triglycerides and glucoselevels were also observed.

Example 8

This example demonstrates that waxes (sterois) were not responsible forthe cholesterol-lowering properties of Louisiana rice bran and its oil.Each group of 6 chickens (6-week old female white leghorn) wasadministered the chick mash diet described in Example 5, supplementedwith one of three different amounts of waxes (sterois), 50 ppm of TRF(prepared as in Example 2) or 5% equivalent of the methanol insolublefraction Louisiana rice bran oil. The amount of feed consumed by allgroups was comparable to the control group and the feeding period was 4weeks. The birds were fasted for a period of 14 hours prior to sacrifice(at 0800 hours). The following protocols were performed on the samplesdisplayed in Table IV:

waxes (entries 2-4): Protocol VII.

TRF (entry 5) was obtained using the method described in Example 2. Themethanol insoluble fraction (entry 6) was obtained by freezing Louisianarice bran oil (prepared using protocols I and IV), then centrifuging.The resultant supernatant was extracted 3 times with twice the volume ofmethanol.

All other samples were obtained from commercially available sources.

The results are displayed below in Table IV. Percentages of increases ordecreases are shown in parentheses.

                                      TABLE IV                                    __________________________________________________________________________                                       HMG-CoA  Cholesterol                                     Serum Cholesterol (mg/100/ml)                                                                      Reductase                                                                              7α-Hydroxylase              Nutritional State.sup.1                                                                     Total Chol.                                                                          HDL-Chol.                                                                            LDL-Chol.                                                                            (pmoles/mg/min).sup.2                                                                  (nmoles/mg/min).sup.3             __________________________________________________________________________    1)                                                                              Chick Diet + 5.0%                                                                         185.1 ± 4.12.sup.A                                                                110.3 ± 4.95.sup.A                                                                61.9 ± 1.49.sup.A                                                                 344.3 ± 1.49.sup.A                                                                  0.855 ± 0.084.sup.A              Corn Oil (CDCO)                                                                           (100.0).sup.4                                                                        (100.0).sup.4                                                                        (100.0).sup.4                                                                        (100.0).sup.4                                                                          (100.0).sup.4                     2)                                                                              Chick Diet + 5.0%                                                                         184.7 ± 6.50.sup.A                                                                109.6 ± 2.83.sup.A                                                                61.7 ± 1.71.sup.A                                                                 339.3 ± 19.7.sup.A                                                                  0.837 ± 0.081.sup.A              Corn Oil + Waxes;                                                                         (99.8) (99.4) (99.7) (98.5)   (97.9)                              50 ppm                                                                      3)                                                                              Chick Diet + 5.0%                                                                         173.8 ± 7.31.sup.A                                                                106.2 ± 4.69.sup.A                                                                58.1 ± 1.77.sup.A                                                                 317.1 ± 14.4.sup.A,B                                                                0.846 ± 0.072.sup.A              Corn Oil + Waxes;                                                                         (93.9) (96.3) (93.9) (92.1)   (98.9)                              5,000 ppm                                                                   4)                                                                              Chick Diet + 5.0%                                                                         165.9 ± 4.90.sup.B                                                                108.5 ± 4.68.sup.A                                                                57.9 ± 1.48.sup.A                                                                 304.5 ± 14.4.sup.B                                                                  0.902 ± 0.080.sup.A              Corn Oil + Waxes;                                                                         (89.6) (98.4) (93.5) (88.4)   (105.5)                             10,000 ppm                                                                  5)                                                                              Chick Diet + 5.0%                                                                         134.8 ± 3.82.sup.C                                                                104.3 ± 3.99.sup.A                                                                25.9 ± 1.02.sup.B                                                                 276.0 ± 17.4.sup.C                                                                  1.068 ± 0.047.sup.B              Corn Oil +  (72.8) (94.6) (41.8) (80.2)   (124.9)                             Tocotrienol-Rich-                                                             Fraction; 50 ppm                                                            6)                                                                              Chick Diet + 5.0%                                                                         180.2 ± 6.01.sup.A                                                                104.0 ± 4.57.sup.A                                                                62.6 ± 1.68.sup.A                                                                 321.1 ± 19.3.sup.B                                                                   .852 ± 0.090.sup.A              Methanol Insoluble                                                                        (97.4) (94.3) (101.1)                                                                              (93.3)   (99.6)                              Fraction                                                                    __________________________________________________________________________     .sup.1 Feeding period was 4 weeks; time of killing was 0800 hours. The        birds were fasted for 14 hours prior to killing. Data expressed as means      ± SD; n = 6 chickens per group.                                            .sup.2 pmoles of mevalonic acid synthesized per minute per mg of              microsomal protein.                                                           .sup.3 nmoles of [.sup.14 C] 7hydroxycholesterol per minute per mg of         microsomal protein.                                                           .sup.4 Percentages of increases or decreases are in parentheses.              .sup.A-D Values not sharing a common superscript letter are different at      < 0.01.                                                                  

The diets supplemented with sterols were not effective in lowering totalserum or LDL-cholesterol levels. The maximum decrease in totalcholesterol recorded with the sterol supplemented diet was 10.6% andthat for LDL-cholesterol was only 6.5%. Only a marginal decrease wasobserved in hepatic enzymatic activity of HMG-CoA reductase (11.6%).Conversely, chickens fed the diet supplemented with the methanol solublefraction of the TRF recorded a substantial decrease in the levels oftotal serum cholesterol (27.2%), LDL-cholesterol (58.2%) and hepaticenzymatic activity of HMG-CoA reductase (19.8%). Therefore, the methanolsoluble fraction of the TRF (containing the tocotrienols according tothis invention), rather than sterols, was responsible for the impressivecholesterol-reducing properties of rice bran oil.

Example 9

This study was conducted to determine if sterols were responsible forthe reduction in serum levels of various enzymes and blood constituentsobserved with rice bran. The feeding conditions described in Example 8were used. The samples were prepared as described in Example 8.

The results are displayed below in Table V. Percentages of increases ordecreases are shown in parentheses.

                                      TABLE V                                     __________________________________________________________________________                  Fatty Acid                      Platelet                                      Synthetase.sup.2                                                                       Triglycerides                                                                        Glucose                                                                              Thromboxane B.sub.2                                                                    Factor 4                        Nutritional State.sup.1                                                                     (pmoles/mg/min)                                                                        (mg/100 ml)                                                                          (mg/100 ml)                                                                          (mg/100 ml)                                                                            (ng/ml)                         __________________________________________________________________________    1)                                                                              Chick Diet + 5.0%                                                                         61.4 ± 2.4.sup.A                                                                    90.2 ± 1.17.sup.A                                                                 124.6 ± 2.3.sup.A                                                                 16.7 ± 1.69.sup.A                                                                   7.2 ± 0.48.sup.A               Corn Oil (CDCO)                                                                           (100.0).sup.3                                                                          (100.0).sup.3                                                                        (100.0).sup.3                                                                        (100.0).sup.3                                                                          (100.0).sup.3                   2)                                                                              Chick Diet + 5.0%                                                                         62.5 ± 1.9.sup.A                                                                    91.5 ± 1.48.sup.A                                                                 126.7 ± 2.1.sup.A                                                                 15.8 ± 1.29.sup.A                                                                   7.5 ± 0.42.sup.A               Corn Oil + Waxes;                                                                         (102.0).sup.3                                                                          (101.4).sup.3                                                                        (101.7).sup.3                                                                        (94.6).sup.3                                                                           (104.2).sup.3                     50 ppm                                                                      3)                                                                              Chick Diet + 5.0%                                                                         63.6 ± 2.8.sup.B                                                                    95.2 ± 1.01.sup.A                                                                 123.9 ± 1.52.sup.A                                                                16.4 ± 1.66.sup.A                                                                   7.4 ± 0.36.sup.A               Corn Oil + Waxes;                                                                         (103.8).sup.3                                                                          (105.5).sup.3                                                                        (99.4).sup.3                                                                         (98.2).sup.3                                                                           (102.8).sup.3                     5,000 ppm                                                                   4)                                                                              Chick Diet + 5.0%                                                                         60.4 ± 1.9.sup.A                                                                    96.1 ± 1.90.sup.A                                                                 124.3 ± 1.18.sup.A                                                                16.8 ± 1.67.sup.A                                                                   7.4 ± 0.87.sup.A               Corn Oil + Waxes;                                                                         (98.5).sup.3                                                                           (106.5).sup.3                                                                        (99.8).sup.3                                                                         (100.6).sup.3                                                                          (102.8).sup.3                     10,000 ppm                                                                  5)                                                                              Chick Diet + 5.0%                                                                         68.5 ± 2.1.sup.B                                                                    73.2 ± 1.69.sup.B                                                                  86.4 ± 1.55.sup.B                                                                12.4 ± 1.42.sup.B                                                                   5.7 ± 0.64.sup.B               Corn Oil +  (111.7).sup.3                                                                          (81.2).sup.3                                                                         (69.3).sup.3                                                                         (74.3).sup.3                                                                           (79.2).sup.3                      Tocotrienol-Rich-                                                             Fraction; 50 ppm                                                            6)                                                                              Chick Diet + 5.0%                                                                         65.8 ± 1.2.sup.B                                                                    89.5 ± 1.21.sup.A                                                                 111.1 ± 2.45.sup.C                                                                15.4 ± 1.64.sup.A                                                                   7.5 ± 0.36.sup.A               Methanol    (107.3).sup.3                                                                          (99.2).sup.3                                                                         (89.4).sup.3                                                                         (92.2).sup.3                                                                           (104.2).sup.3                     Insoluble                                                                     Fraction                                                                    __________________________________________________________________________     .sup.1 Feeding period was 4 weeks; time of killing was 0800 hours. The        birds were fasted for 14 hours prior to killing. Data expressed as means      ± SD; n = 6 chickens per group.                                            .sup.2 nmoles of NADPH oxidized per minute per mg of cytosolic protein.       .sup.3 Percentages of increases or decreases are in parentheses.              .sup.A-C Values not sharing a common superscript letter are different at      < 0.01.                                                                  

These results show that the sterols are not responsible for thereduction in the levels of triglycerides, glucose, thromboxane B₂ orplatelet factor 4 levels observed with rice bran and its oil. In fact,the sterols either increased or had no significant effect on the levelof each of those factors. Conversely, the chickens fed with the dietsupplemented with the methanol soluble fraction of the TRF of Louisianarice bran induced significant decreases in all of these factors.

Example 10

This study measured the effects of three tocotrienols isolated from ricebran on cholesterol-related enzyme activity in hepatocytes isolated fromlivers of 8-week old female chickens. The chickens were fed the chickmash diet for 8 weeks. They were then fasted for 40 hours and finallyrefed for 48 hours before sacrifice. The hepatocytes were then preparedfollowing standard methods.

Each compound was isolated from TRF using the following protocol: A bondElute amine column was equilibrated with 2 ml hexane. 10 mg of TRF(dissolved in 0.5 ml hexane) was bound to the amine column. The columnwas washed with 1 ml of hexane, then with 1 ml of 3% isopropanol inhexane. P-21 and P-25 were eluted off the column with 5% isopropanol inhexane. Then using 10% isopropanol in hexane, the impurity P-20 waseluted. α-tocotrienol was also tested.

The results are displayed below in Table VI. Percentages of increases ordecreases are shown in parentheses.

                  TABLE VI                                                        ______________________________________                                        Con-                                                                          centration                                                                            HMG-CoA     Fatty Acid  Cholesterol 7α-                         in.sup.1                                                                              Reductase   Synthase    hydroxylase                                   (μg/ml)                                                                            pmoles/min/mg                                                                             nmoles/min/mg                                                                             pmoles/min/mg                                 ______________________________________                                        A) α-Tocotrienol                                                        1.   0.0    26.8    (100.0)                                                                             17.2  (100.0)                                                                             2.31  (100.0)                           2.   10.0   21.4     (79.9)                                                                             15.2   (88.4)                                                                             2.12   (91.8)                           3.   20.0   17.3     (64.6)                                                                             14.3   (83.2)                                                                             2.27   (98.7)                           4.   40.0   16.7     (62.3)                                                                             12.4   (72.1)                                                                             2.34  (101.3)                           B) P-21 (Tocotrienol)                                                         1.   0.0    26.8    (100.0)                                                                             17.2  (100.0)                                                                             2.31  (100.0)                           2.   10.0   20.4     (76.1)                                                                             14.3   (83.1)                                                                             2.41  (104.3)                           3.   20.0   16.7     (62.3)                                                                             12.7   (73.8)                                                                             3.40  (103.9)                           4.   40.0   14.2     (52.9)                                                                             11.3   (68.6)                                                                             2.39  (103.5)                           5.   80.0   14.3     (53.4)                                                                             10.2   (59.3)                                                                             1.37  (102.6)                           C) P-25                                                                       1.   0.0    26.5    (100.0)                                                                             17.2  (100.0)                                                                             2.31  (100.0)                           2.   10.0   19.0     (70.9)                                                                             16.2   (94.2)                                                                             2.33  (100.9)                           3.   20.0   15.3     (57.1)                                                                             14.3   (83.1)                                                                             2.37  (102.61)                          4.   40.0   12.4     (46.3)                                                                             12.7   (73.8)                                                                             2.44  (105.6)                           5.   80.0   11.1     (41.4)                                                                             12.1   (70.3)                                                                             2.37  (102.6)                           D) P-20                                                                       1.   0.0    28.78   (100.0)                                                   2.   25.0   29.21   (101.5)                                                   3.   55.0   29.87   (100.3)                                                   4.   100.0  28.21    (98.0)                                                   ______________________________________                                         .sup.1 Feeding period was 8 weeks; the birds were fasted for 40 hrs. and      refed for 48 hrs. The hepatocytes were prepared at 10 PM from two livers      by standard methods.                                                     

P₂₅ demonstrated very effective dose dependant inhibition of HMG-CoAreductase with maximum reductions in activity of 46.6% and 58.6%,respectively. In fact, P₂₅ exerted substantially better effects than theknown hypercholesterolemic agent, α-tocotrienol. α-tocotrienol showed amaximum reduction of only 37.7%. P₂₀, the sterol contaminant, showed nosignificant effect on the activity of HMG-CoA reductase.

Example 11

We next measured the effects of various tocotrienols andtocotrienol-like compounds isolated from rice bran on the activity ofHMG-CoA reductase. 20.0 g of stabilized rice bran was extracted with200.0 ml of methanol to remove various UV absorbing impurities andtocotrienols (6.7 mg total). This step was repeated four times. Theremaining residue was dried under vacuum in a desiccator and then heatedat 80° C. for 2 hrs under 30 psi pressure. Then the dried residue wasextracted again with 200.0 ml methanol (3.4 mg). The various peaks werepurified by HPLC except 100 μl was injected instead of 20 μl. Twentyruns were done to obtain enough material for this study. The peaks whicheluted at 32, 36, 45 and 54 minutes were tested in chicken hepatocytesas in Example 10.

The results of the HMG-CoA reductase assay are displayed below in TableVII.

                  TABLE VII                                                       ______________________________________                                                   NMG-CoA Reductase                                                  Concentration.sup.1                                                                        (pmoles/min/mg of microsome)                                     (μg/ml)   P-32   P-36       P-45 P-54                                      ______________________________________                                        1)     0.00      28.78  28.78    28.78                                                                              28.78                                   2)    10.00      24.67  23.75    22.61                                                                              25.46                                   3)    20.00      21.31  20.12    20.33                                                                              19.67                                   4)    30.00      18.36  17.24    19.67                                                                              20.11                                   5)    40.00      16.59  17.34    18.21                                                                              18.19                                   6)    50.00      16.61  15.21    18.40                                                                              18.20                                   ______________________________________                                         .sup.1 Feeding period was 8 weeks; the birds were fasted for 40 hrs. and      refed for 48 hrs. The hepatocytes were prepared at 10 PM from two livers      by standard methods.                                                     

Each of these compounds demonstrated an ability to inhibit the activityof HMG-CoA reductase. Most notably, P₃₆ showed a 47% maximum reductionof activity.

Example 12

This example measured the effects of the TRF and its components on thehepatic enzymatic activity of HMG-CoA reductase and cholesterol7α-hydroxylase. Each group of 12 chickens (6-week old female whiteleghorn) was administered either a control diet or a control dietsupplemented with the TRF, a component of the TRF a commercialcholesterol inhibitor (either Lovastatin or Geraniol) or a combinationof the commercial inhibitors. The amount of feed consumed ranged from11.19-11.60 g per chicken and the feeding period was 4 weeks. The birdswere fasted for a period of 36 hours and refed for 48 hours prior tosacrifice (at 0800 hours).

The TRF (entry 2) was prepared as described in Example 10. Geraniol wasobtained from Sigma Chemical Co. and Lovastatin was obtained under thebrand name LOVOCOR.

The chicken diet for this study contained the following ingredients:

    ______________________________________                                        Ingredients       Percentage                                                  ______________________________________                                        Corn (9.3% protein)                                                                             61.5                                                        Soybean Meal      30.0                                                        (44.0% protein)                                                               Meat scrap        5.0                                                         (50.0% protein)                                                               Calcium Carbonate 0.5                                                         Dicalcium Phosphate                                                                             1.0                                                         Alfalfa (17% protein)                                                                           1.0                                                         Mineral Mixture.sup.a                                                                           0.5                                                         Vitamin Mixture.sup.b                                                                           0.5                                                         ______________________________________                                         .sup.a Mineral mixture contained per kg feed: zinc sulfate, 50 mg; sodium     chloride, 2.0 mg; and manganese dioxide, 50.0 mg.                             .sup.b Vitamin mixture contained per kg feed: vitamin A, 2,000 units;         vitamin D.sub.3, 200 units; vitamin E, 10 units, riboflavin, 3.6 mg;          vitamin B.sub.12, 0.01 mg; and vitamin K.sub.1, 0.50 mg.                 

The results are displayed below in Table VIII. Percentages of increasesor decreases are in parentheses.

                  TABLE VIII                                                      ______________________________________                                                        HMG-CoA     Cholesterol                                                       Reductase.sup.2                                                                           7α-Hydroxylase.sup.3                        Nutritional State.sup.1                                                                       pmole/min./mg                                                                             nmole/min./mg                                     ______________________________________                                        1)  Control Diet (CD)                                                                             513.26 ± 15.07.sup.A                                                                   10.22 ± 0.25.sup.A                                             (100.00).sup.4                                                                            (100.00).sup.4                                2)  CD + TRF-RBO;   441.14 ± 7.28.sup.B                                                                    10.80 ± 0.18.sup.A                             50 ppm          (85.95).sup.4                                                                             (105.00).sup.4                                3)  CD + α - T.sub.3 ; 50 ppm                                                               459.02 ± 1505.sup.B                                                                    10.69 ± 0.20.sup.A                                             (89.43).sup.4                                                                             (104.60).sup.4                                4)  CD + γ - T.sub.3 ; 50 ppm                                                               401.99 ± 5.84.sup.C                                                                    10.97 ± 0.17.sup.A                                             (78.32).sup.4                                                                             (107.34).sup.4                                5)  CD + δ - T.sub.3 ; 50 ppm                                                               389.22 ± 8.54.sup.C                                                                    10.98 ± 0.16.sup.A                                             (74.83).sup.4                                                                             (107.44).sup.4                                6)  CD + P-21 - T.sub.3 ;                                                                         373.78 ± 7.99.sup.D                                                                    11.49 ± 0.20.sup.B                             50 ppm          (72.83).sup.4                                                                             (12.43).sup.4                                 7)  CD + P-25 - T.sub.3 ;                                                                         366.10 ± 6.66.sup.D                                                                    11.39 ± 0.26.sup.B                             50 ppm          (71.33).sup.4                                                                             (111.45).sup.4                                8)  CD + Geraniol;  449.89 ± 12.82.sup.B                                                                   10.28 ± 0.27.sup.A                             100 ppm         (87.65).sup.4                                                                             (100.59).sup.4                                9)  CD + Lovastatin;                                                                              527.11 ± 13.93.sup.A                                                                   11.34 ± 0.31.sup.B                             100 ppm         (102.69).sup.4                                                                            (110.96).sup.4                                10) CD + Geraniol + 442.48 ± 1.64.sup.B                                                                    11.24 ± 0.17.sup.B                             Lovastatin 50 ppm +                                                                           (86.21).sup.4                                                                             (109.98).sup.4                                    50 ppm                                                                    ______________________________________                                         .sup.1 Feeding period was 4 weeks. Time of sacrificing was 0800 hr. The       birds were fasted for 36 hrs. and refed 48 hrs. at the end of feeding         period (28 days), and then sacrificed. Data expressed as means ± SD; n     = 12 birds per group.                                                         .sup.2 pmoles of HMGCoA formed per minute per mg of microsomal protein.       .sup.3 nmoles of [.sup.14 ccholesterol into [.sup.14c] 7hydroxycholestero     per minute per mg of microsomal protein.                                      .sup.4 Percentages of increases are in parentheses.                           .sup.A-D Values not sharing a common superscript letter are different at      < 0.01.                                                                  

P₂₅ was the best inhibitor of HMG-CoA reductase. The enzymatic activityof HMG-CoA reductase was reduced by about 28% with P₂₅, while twice theamount of the commercial cholesterol-lowering drugs provided a maximumreduction of only about 13%. These data indicate that the fused oxygenheterocycle of the tocotrienols increases the biological activity ascompared with a single ring system, such as Geraniol. We believe that athree ring system might provide additional activity. Alternatively,adding substituents such as carbonyls, hydroxyls and alkyls may alsoenhance the biological activity of tocotrienol-like compounds.

Example 13

We next studied the effects of the TRF and its components on total serumcholesterol levels, the HDL-cholesterol/total cholesterol ratio and theHDL-/LDL-chlolesterol ratio. Feeding conditions were identical to thosein Example 12.

The samples were prepared as described in Example 12.

The results are displayed below in Table IX. Percentages of increases ordecreases are in parentheses.

                                      TABLE IX                                    __________________________________________________________________________                Concentration in serum (mg/dl)                                                 Total   HDL-   LDL-   HDL-Cholesterol/                                                                        HDL-Cholesterol/                 NUTRITIONAL STATE.sup.1                                                                    Cholesterol                                                                           Cholesterol                                                                          Cholesterol                                                                          Total Cholesterol                                                                       LDL-Cholesterol                  __________________________________________________________________________    1)                                                                              Control Diet (CD)                                                                        142.77 ± 1.916.sup.A                                                               83.66 ± 0.85.sup.A                                                                55.06 ± 0.89.sup.A                                                                0.59      1.52                                          (100.00).sup.2                                                                        (100.00).sup.2                                                                       (100.00).sup.2                                                                       (100.00).sup.2                                                                          (100.00).sup.2                   2)                                                                              CD + TRF - RBO                                                                           125.10 ± 1.50.sup.B                                                                78.71 ± 1.06.sup.B                                                                43.47 ± 0.78.sup.B                                                                0.63      1.81                               50 ppm     (87.62) (94.08)                                                                              (78.95)                                                                              (102.40)  (119.20)                         3)                                                                              CD + α - T.sub.3                                                                   131.71 ± 1.08.sup.C                                                                80.47 ± 0.67.sup.B                                                                47.97 ± 0.56.sup.C                                                                0.61      1.68                               50 ppm     (92.25) (96.19)                                                                              (87.12)                                                                              (104.30)  (110.40)                         4)                                                                              CD + γ - T.sub.3                                                                   116.86 ± 1.47.sup.D                                                                78.39 ± 0.96.sup.B                                                                37.52 ± 0.81.sup.D                                                                0.67      2.09                               50 ppm     (81.85) (93.70)                                                                              (68.14)                                                                              (114.50)  (137.50)                         5)                                                                              CD + δ - T.sub.3                                                                   119.40 ± 1.41.sup.D                                                                77.87 ± 0.97.sup.B                                                                36.14 ± 0.97.sup.D                                                                0.65      2.15                               50 ppm     (83.63) (93.08)                                                                              (65.64)                                                                              (111.30)  (141.80)                         6)                                                                              CD + P-21 - T.sub.3                                                                       92.91 ± 1.27.sup.E                                                                68.88 ± 1.52.sup.C                                                                22.16 ± 0.57.sup.E                                                                0.74      3.11                               50 ppm     (65.08) (82.33)                                                                              (40.25)                                                                              (126.50)  (204.60)                         7)                                                                              CD + P-25 - T.sub.3                                                                       95.51 ± 1.11.sup.E                                                                70.12 ± 1.25.sup.C                                                                23.00 ± 1.27.sup.E                                                                0.73      3.05                               50 ppm     (66.90) (83.82)                                                                              (41.77)                                                                              (125.30)  (200.60)                         8)                                                                              CD + Geraniol                                                                            122.53 ± 1.37.sup.D                                                                82.28 ± 1.06.sup.A                                                                41.07 ± 1.13.sup.F                                                                0.65      2.00                               100 ppm    (85.82) (98.35)                                                                              (96.30)                                                                              (110.10)  (131.50)                         9)                                                                              CD + Lovastatin                                                                          128.65 ± 1.02.sup.C                                                                81.38 ± 0.73.sup.A                                                                44.12 ± 1.26.sup.B                                                                0.63      1.84                               100 ppm    (90.11) (97.28)                                                                              (86.51)                                                                              (108.00)  (121.40)                         10)                                                                             CD + Geraniol +                                                                          119.92 ± 0.91.sup.D                                                                80.93 ± 0.62.sup.B                                                                41.31 ± 1.13.sup.B                                                                0.67      1.96                               Lovastatin;                                                                              (84.00) (96.74)                                                                              (89.29)                                                                              (115.20)  (128.90)                           50 ppm + 50 ppm                                                             __________________________________________________________________________     .sup.1 Feeding period was 4 weeks. Time of drawing the blood was 0800 hr.     Data expressed as means ± SD; n = 12 birds per group.                      .sup.2 Percentages of increases or decreases are in parentheses.              .sup.A-F Values not sharing common superscript letter re different at P <     0.01.                                                                    

P₂₁ and P₂₅ decreased the levels of serum cholesterol to a substantiallygreater degree than any other compound tested, including Lovastatin andGeraniol. P₂₅ caused about a 33% reduction. In comparison, Geranioleffected a 14% reduction and Lovastatin caused only a 10% reduction. P₂₁and P₂₅ were also clearly superior than the other compounds inincreasing the HDL-/LDL-cholesterol ratio. Both of these compoundsinduced over a 100% increase in the HDL-/LDL-cholesterol ratio, whileLovastatin and Geraniol increased the ratio by a maximum of 31.5%.

Example 14

We next measured the effects of TRF and its components on serum levelsof apolipoprotein (a)₁, apolipoprotein B, triglycerides and glucose andthe plasma levels of thromboxane B₂ and platelet factor 4. Feedingconditions were identical to Example 12.

The samples were prepared as described in Example 12.

The results are displayed below in Table X. Percentages of increases ordecreases are in parentheses.

                                      TABLE X                                     __________________________________________________________________________                                                      Platelet                                 Concentration In Serum (mg/100 ml)                                                                         Thromboxane                                                                           Factor 4                    Nutritional State.sup.1                                                                    APO A.sub.1                                                                          APO B  Triglycerides                                                                        Glucose B.sub.2 (pg/ml)                                                                       (ng/ml)                     __________________________________________________________________________    1)                                                                              Control diet (CD)                                                                        140.40 ± 1.7.sup.A                                                                33.10 ± 0.28.sup.A                                                                74.03 ± 1.26.sup.A                                                                224.30 ± 5.30.sup.A                                                                25.84 ± 0.84.sup.A                                                                 13.09 ± 0.49.sup.A                    (100.00).sup.2                                                                       (100.00).sup.2                                                                       (100.00).sup.2                                                                       (100.00).sup.2                                                                        (100.00).sup.2                                                                        (100.00).sup.2              2)                                                                              CD + TRF - RBO;                                                                          140.20 ± .8.sup.A                                                                 28.90 ± 0.28.sup.B                                                                64.73 ± 0.93.sup.B                                                                212.50 ± 0.98.sup.B                                                                20.55 ± 0.95.sup.B                                                                  9.67 ± 0.43.sup.B         50 ppm     (99.86)                                                                              (87.31)                                                                              (87.44)                                                                              (94.74) (79.53) (73.87)                     3)                                                                              CD + α - T.sub.3 ;                                                                 139.60 ± 3.2.sup.A                                                                29.30 ± 0.16.sup.B                                                                68.04 ± 1.32.sup.C                                                                216.90 ± 1.62.sup.B                                                                21.63 ± 0.89.sup.B                                                                  8.78 ± 0.72.sup.C         50 ppm     (99.43)                                                                              (88.52)                                                                              (91.91)                                                                              (96.70) (83.71) (67.07)                     4)                                                                              CD + γ - T.sub.3 ;                                                                 139.80 ± 1.5.sup.A                                                                29.53 + 3.47.sup.B                                                                   61.07 ± 1.37.sup.D                                                                208.70 ± 1.41.sup.C                                                                19.22 ± 0.78.sup.B,C                                                                8.36 ± 0.79.sup.C         50 ppm     (99.57)                                                                              (89.21)                                                                              (82.49)                                                                              (93.05) (74.38) (63.87)                     5)                                                                              CD + δ - T.sub.3 ;                                                                 139.30 ± 1.4.sup.A                                                                27.30 ± 0.28.sup.B                                                                60.50 ± 2.50.sup.D                                                                205.30 ± 1.83.sup.C                                                                18.65 + 0.99.sup.C                                                                     8.23 ± 0.77.sup.C         50 ppm     (99.22)                                                                              (82.48)                                                                              (81.72)                                                                              (91.53) (72.17) (62.87)                     6)                                                                              CD + P-21 - T.sub.3 ;                                                                    138.40 ± 4.4.sup.A                                                                25.40 ± 0.40.sup.C                                                                56.22 ± 1.11.sup.E                                                                198.80 ± 2.10.sup.D                                                                16.74 ± 1.62.sup.C                                                                  7.76 ± 1.67.sup.C         50 ppm     (98.79)                                                                              (76.74)                                                                              (75.94)                                                                              (88.63) (64.78) (59.28)                     7)                                                                              CD + P-25 - T.sub.3 ;                                                                    138.70 ± 2.7.sup.A                                                                25.20 ± 0.40.sup.C                                                                54.65 ± 1.42.sup.E                                                                196.10 ± 1.74.sup.D                                                                16.42 ± 1.36.sup.C                                                                  7.27 ± 0.74.sup.C         50 ppm     (98.78)                                                                              (76.13)                                                                              (73.82)                                                                              (87.43) (63.54) (55.54)                     8)                                                                              CD + Geraniol;                                                                           138.40 ± 1.7.sup.A                                                                29.20 ± 0.45.sup.B                                                                71.29 ± 1.19.sup.F                                                                227.60 ± 2.03.sup.A                                                                25.46 ± 1.52.sup.A                                                                 12.38 ± 1.26.sup.B         100 ppm    (98.58)                                                                              (88.22)                                                                              (96.30)                                                                              (101.47)                                                                              (98.53) (94.57)                     9)                                                                              CD + Lovastatin;                                                                         139.90 1.4.sup.D                                                                     25.80 ± 0.89.sup.C                                                                64.04 ± 1.03.sup.B                                                                220.10 + 1.32.sup.D                                                                   24.89 ± 0.88.sup.A                                                                 12.24 ± 1.49.sup.B         100 ppm    (99.29)                                                                              (77.95)                                                                              (86.51)                                                                              (98.13) (96.32) (93.51)                     10)                                                                             CD + Geraniol +                                                                          139.60 ± 4.8.sup.A                                                                25.95 ± 0.34.sup.C                                                                66.10 ± 0.99.sup.B                                                                221.70 ± 1.89.sup.D                                                                24.95 ± 0.97.sup.A                                                                 12.19 ± 1.15.sup.B         Lovastatin;                                                                              (99.43)                                                                              (78.40)                                                                              (89.29)                                                                              (98.84) (96.56) (93.12)                       50 ppm + 50 ppm                                                             __________________________________________________________________________     .sup.1 Feeding period was 4 weeks. Time of drawing the blood was 0800 hr.     Data expressed as means ± SD; n = 12 birds per group.                      .sup.2 Percentages of increasees or decreases are in parentheses.             .sup.A-F Values not sharing a common superscript letter are different at      < 0.01.                                                                  

P₂₁ and P₂₅ were the most potent cholesterol inhibitors. They effectedthe highest Apo A₁ to Apo B ratio and resulted in the maximum decreasein serum levels of triglycerides and glucose. In addition, they alsoreduced plasma levels of thromboxane B₂ and platelet factor 4 to agreater extent than any other compound tested.

Example 15

We next measured the effects of various tocotrienols from rice bran onHMG-CoA reductase and cholesterol 7α-hydroxylase in hypercholesterolemicswine. Since pigs are very similar to humans in their cholesterolmetabolism, they provide a useful model from which to studyhypercholesterolemia and related diseases. The pigs used in this studycarried Lpd⁵ and Lpu¹ mutant alleles for apolipoprotein B and U. Becauseof this genetic defect, these pigs demonstrate spontaneously elevatedLDL-cholesterol levels and hypercholesterolemia. The serum cholesterolconcentration in these swine is typically above 300-500 mg/dl, ascompared to 120-160 mg/dl in normal adult swine. As a result, the pigsdevelop complicated atherosclerotic plaques that closely resembleadvanced atherosclerotic lesions found in humans.

Each group of three 5-month old swine was administered either a controldiet or a control diet supplemented with 50 ppm of TRF or 50 ppm of anindividual tocotrienol. The samples were prepared as described inExample 11. After a 12 hour fast, serum and plasma samples were taken at0 hrs, 21 days and 42 days from the start of the feeding period. Aftertaking serum and plasma samples from the pigs at the end of the 42 dayperiod, all of the swine were fasted for a total of 40 hours, followedby a 48 hour refeeding period. One swine from each group was sacrificedat that time and the liver, intestine, lung, heart, loin muscle, adiposetissue and ham muscle were removed.

The swine diet contained the following ingredients:

    ______________________________________                                        Ingredients       Percentage                                                  ______________________________________                                        Corn (9.3% protein)                                                                             78.37                                                       Soybean Meal      15.42                                                       (44.0% protein)                                                               Lard              3.00                                                        Calcium Carbonate 0.95                                                        Dicalcium Phosphate                                                                             0.96                                                        Mineral Mixture.sup.a                                                                           0.30                                                        Vitamin Mixture.sup.b                                                                           1.00                                                        ______________________________________                                         .sup.a Mineral mixture contained per kg feed: zinc sulfate H.sub.2 O, 110     mg; manganese sulfate 5H.sub.2 O, 70 mg; ferric citrate H.sub.2 O, 500.0      mg; copper sulfate 5H.sub.2 O, 16.0 mg; sodium selenite, 0.2 mg;              DLmethionine, 2.5 g; choline chloride (50%), 1.5 g; ethoxyguin                (1,2dihydro-6-ethoxy-2,2,4-trimethyl-quinoline), 125 mg; and thiamineHCl,     1.8 mg.                                                                       .sup.b Vitamin mixture contained per kg feed: vitamin A, 1,500 units;         vitamin D.sub.3, 400 units; vitamin E, 10 units, riboflavin, 3.6 mg;          calcium pantothenate, 10.0 mg; niacin, 25.0 mg; pyridoxineHCl, 3.0 mg;        folacin, 0.55 mg; biotin, 0.15 mg; vitamin B.sub.12, 0.01 mg; and vitamin     K.sub.1, 0.55 mg.                                                        

The gain in body weight in all groups was comparable to the control,although feed conversion efficiency increased 7% with P₂₁ and 10% withP₂₅.

The results of this study are displayed in Table XI. Percentages ofincreases and decreases are shown in parentheses.

                                      TABLE XI                                    __________________________________________________________________________    HMG-CoA REDUCTASE.sup.2 pg/min/mg of microsomes                                              Experimental Diets      Percentage                                            Feeding period          of Control                             NUTRITIONAL STATE.sup.1                                                                      0-Time  21 days 42 days Activity                               __________________________________________________________________________    Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                         355.49 ± 8.44                                                                      374.14 ± 13.35                                                                     391.38 ± 15.75                                                                     100.00.sup.A                                          (100.00 (105.25)                                                                              (110.09)                                       3) CD + TRF RB-oil; 50 ppm                                                                   348.92 ± 10.82                                                                     300.72 ± 10.53                                                                     285.13 ± 15.58                                                                     74.23.sup.A                                           (100.00)                                                                              (86.19) (81.72)                                        4) CD + γ - T.sub.3 ; 50 ppm                                                           347.99 ± 16.29                                                                     300.72 ± 10.53                                                                     280.88 ± 12.81                                                                     73.31.sup.B                                           (100.00)                                                                              (86.19) (80.71)                                        5) CD + P-21 - T.sub.3 ; 50 ppm                                                              340.36 ± 23.55                                                                     304.23 ± 23.13                                                                     275.75 ± 12.76                                                                     73.59.sup.B                                           (100.00)                                                                              (89.38) (81.02)                                        6) CD + P-25 - T.sub.3 ; 40 ppm                                                              350.86 ± 23.79                                                                     307.27 ± 23.36                                                                     269.43 ± 13.23                                                                     69.75.sup.B                                           (100.00)                                                                              (87.58) (76.79)                                        __________________________________________________________________________     .sup.1 Cornsoybean meal control diet or experimental diets were fed to        5month-old swine for 42 days. Then all groups were fasted for 40 hrs. and     refed for 48 days. The time of sacrificing was 9:00 a.m. All tissues were     kept on ice. TRF = TocotrienolRich-Fraction.                                  .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group;                                                                        .sup.A-B Values not sharing a common superscript letter are different at      < 0.01.                                                                  

    __________________________________________________________________________    CHOLESTEROL 7α-HYDROXYLASE.sup.2 pg/min/mg of                           microsomes                                                                    Experimental Diets               Percentage                                   Feeding Period                   of Control                                   NUTRITIONAL STATE.sup.1                                                                      0-Time                                                                              21 days                                                                             42 days                                                                             Activity                                     __________________________________________________________________________    Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                         4.88 ± 0.75                                                                      5.00 ± 0.60                                                                      5.07 ± 0.61                                                                      100.00.sup.A                                                (100.00).sup.A                                                                      (102.46).sup.3                                                                      (103.89).sup.3                                     3) CD + TRF RB-oil; 50 ppm                                                                   4.58 ± 0.10                                                                      4.96 ± 0.13                                                                      5.02 ± 0.11                                                                      105.50.sup.A                                                (100.00)                                                                            (108.29)                                                                            (109.61)                                           4) CD + γ - T.sub.3 ; 50 ppm                                                           4.49 ± 0.13                                                                      4.53 ± 0.12                                                                      4.58 ± 0.19                                                                      98.18.sup.A                                                 (100.00)                                                                            (100.89)                                                                            (102.00)                                           5) CD + Desmethyl - T.sub.3 ;                                                                4.53 ± 0.10                                                                      4.50 ± 0.18                                                                      4.63 ± 0.56                                                                      989.38.sup.A                                   (P-21); 50 ppm                                                                             (100.00)                                                                            (99.34)                                                                             (102.21)                                           6) CD + Didesmethyl - T.sub.3 ;                                                              4.57 ± 0.19                                                                      4.62 ± 0.19                                                                      4.68 ± 0.18                                                                      98.58.sup.A                                    (P-25); 50 ppm                                                                             (100.00)                                                                            (101.09)                                                                            (102.41)                                           __________________________________________________________________________     .sup.1 Cornsoybean m eal control diet or experimental diets were fed to       5month-old swine for 42 days. Then all groups were fasted for 40 hrs. and     refed for 48 days. The time of sacrificing was 9:00 a.m. All tissues were     kept on ice. TRF = TocotrienolRich-Fraction.                                  .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group;                                                                        .sup.3 Percentages of increases or decreases in respect to baseline value     .sup.A-B Values not sharing a common superscript letter are different as      < 0.01.                                                                  

The hepatic enzymatic activity of the HMG-CoA reductase decreasedapproximately 30% with all the tocotrienols tested. In addition, therewas no significant decrease in the activity of cholesterol7α-hydroxylase. These results were all time dependent and point to anoverall reduction in cholesterogenesis, with no increase in cholesterolbiosynthesis.

Example 16

Further tests were conducted on the swine described in Example 16. Theexperimental conditions were the same, except that the two remainingswine in each group were transferred to the unsupplemented control dietfollowing the 42 day feeding period. After an additional 70 days, serumand plasma samples were collected from these swine after a 12 hour fastfor testing.

The samples were prepared as described in Example 12.

The results of these studies are displayed below in Tables XII-XV.Percentages of increases and decreases are shown in parentheses.

                                      TABLE XII                                   __________________________________________________________________________    TOTAL CHOLESTEROL CONCENTRATION IN SERUM (mg/100 ml).sup.2                                  Experimental Diets      Percentage                                                                          Control Diet                                                                          Percentage                              Feeding Period          of Control                                                                          Feeding Period                                                                        of Control                NUTRITIONAL STATE.sup.1                                                                     0-Time  21 days 42 days Activity                                                                            70 days Activity                  __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                        116.53 ± 5.64                                                                      131.83 ± 6.64                                                                      142.50 ± 2.75                                                                      --    147.20 ± 4.45                                                                      --                                      (100.00).sup.3                                                                        (113.13).sup.3                                                                        (122.29).sup.3                                                                              (126.32).sup.3                    Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        228.63 ± 221.89                                                                    251.07 ± 16.82                                                                     356.87 ± 8.69                                                                      100.00.sup.A                                                                        374.36 ± 13.50                                                                     100.00.sup.A                            (100.00)                                                                              (110.02)                                                                              (156.79)      (164.60)                          3) CD + TRF RD-oil;                                                                         206.43 ± 18.45                                                                     222.00 ± 15.75                                                                     243.03 ± 12.28                                                                     75.00.sup.B                                                                         285.77 ± 14.00                                                                     84.60.sup.B                 50 ppm      (100.00)                                                                              (107.39)                                                                              (117.72)      (139.16)                          4) CD + γ T.sub.3 ; 50 ppm                                                            209.73 ± 8.50                                                                      225.20 ± 8.36                                                                      233.93 ± 5.35                                                                      71.00.sup.B                                                                         271.70 ± 6.84                                                                      78.80.sup.B                             (100.00)                                                                              (107.39)                                                                              (111.76)      (129.66)                          5) CD + P-21 - T.sub.3 ;                                                                    220.50 ± 16.54                                                                     236.40 ± 18.17                                                                     238.17 ± 14.29                                                                     68.90.sup.B                                                                         276.23 ± 15.93                                                                     76.20.sup.B                 50 ppm      (100.00)                                                                              (107.20)                                                                              (108.20)      (125.39)                          6) CD + P-25 - T.sub.3 ;                                                                    216.20 ± 12.50                                                                     229.90 ± 13.979                                                                    237.64 ± 12.15                                                                     68.60.sup.B                                                                         268.90 ± 11.41                                                                     75.60.sup.B                 50 ppm      (100.00)                                                                              (106.33)                                                                              (107.60)      (124.45)                          __________________________________________________________________________     .sup.1 Feeding period was 6 weeks. Time of drawing the blood was 0.009 hr     after fsted for 12 hrs.; TRF = TocotrienolRich-Fraction.                      .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group.                                                                        .sup.3 Percentages of increases or decreases in respect to baseline value     .sup.A-B Values not sharing a common superscript letter are different at      < 0.01.                                                                  

                                      TABLE XIII                                  __________________________________________________________________________    HDL-CHOLESTEROL CONCENTRATION IN SERUM (mg/100 ml).sup.2                                    Experimental Diets      Percentage                                                                          Control Diet                                                                          Percentage                              Feeding Period          of Control                                                                          Feeding Period                                                                        of Control                NUTRITIONAL STATE.sup.1                                                                     0-Time  21 days 42 days Activity                                                                            70 days Activity                  __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                        20.76 ± 0.67                                                                       23.63 ± 0.62                                                                       28.41 ± 1.67                                                                       --    29.78 ± 1.43                                                                       --                                      (100.00).sup.3                                                                        (113.82).sup.3                                                                        (136.85).sup.3                                                                              (143.45).sup.3                    Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        24.04 ± 1.53                                                                       25.82 ± 0.86                                                                       28.27 ± 0.69                                                                       100.00.sup.A                                                                        31.42 ± 1.67                                                                       100.00.sup.A                            (100.00)                                                                              (107.40)                                                                              (117.59)      (130.69)                          3) CD + TRF RD-oil;                                                                         23.36 ± 1.35                                                                       25.14 ± 1.49                                                                       26.50 ± 1.84                                                                       96.47.sup.A                                                                         30.19 ± 1.21                                                                       98.89.sup.A                 50 ppm      (100.00)                                                                              (107.62)                                                                              (113.44)      (129.24)                          4) CD + γ - T.sub.3 ; 50 ppm                                                          21.86 ± 1.67                                                                       25.27 ± 1.62                                                                       28.55 ± 1.62                                                                       111.06.sup.B                                                                        30.47 ± 1.62                                                                       106.66.sup.A                            (100.00)                                                                              (115.59)                                                                              (130.60)      (139.39)                          5) CD + P-21 - T.sub.3 ;                                                                    22.95 ± 1.73                                                                       26.23 ± 0.89                                                                       30.74 ± 1.13                                                                       113.90.sup.B                                                                        31.15 ± 1.73                                                                       103.80.sup.A                50 ppm      (100.00)                                                                              (114.29)                                                                              (133.94)      (135.72)                          6) CD + P-25 - T.sub.3 ;                                                                    21.86 ± 0.99                                                                       25.68 ± 1.67                                                                       31.81 ± 1.75                                                                       123.75.sup.B                                                                        32.65 ± 1.09                                                                       114.29.sup.A                50 ppm      (100.00)                                                                              (117.47)                                                                              (145.52)      (149.36)                          __________________________________________________________________________     .sup.1 Feeding period was 6 weeks. Time of drawing the blood was 0.009 hr     after fasting for 12 hrs.                                                     .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group; TRF = TocotrienolRich-Fraction.                                        .sup.3 Percentages of increases or decreases in respect to baseline value     .sup.A-B Values not sharing a common superscript letter are different at      < 0.01.                                                                  

                                      TABLE XIV                                   __________________________________________________________________________    LDL-CHOLESTEROL CONCENTRATION IN SERUM (mg/100 ml).sup.2                                    Experimental Diets      Percentage                                                                          Control Diet                                                                          Percentage                              Feeding Period          of Control                                                                          Feeding Period                                                                        of Control                NUTRITIONAL STATE.sup.1                                                                     0-Time  21 days 42 days Activity                                                                            70 days Activity                  __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                         92.84 ± 1.01                                                                      107.31 ± 1.22                                                                      110.88 ± 9.66                                                                      --    115.70 ± 11.69                                                                     --                                      (100.00).sup.3                                                                        (115.58).sup.3                                                                        (119.43).sup.3                                                                              (124.62).sup.3                    Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        192.84 ± 11.89                                                                     216.39 ± 13.46                                                                     319.39 ± 6.02                                                                      100.00.sup.A                                                                        274.11  100.00.sup.A                            (100.00)                                                                              (112.21)                                                                              (164.07)      (142.14)                          3) CD + TRF RD-oil;                                                                         177.28 ± 10.97                                                                     187.15 ± 13.49                                                                     209.09 ± 11.86                                                                     71.88.sup.B                                                                         267.08 ± 7.7                                                                       105.99.sup.A                50 ppm      (100.00)                                                                              (105.57)                                                                              (117.94)      (150.65)                          4) CD + γ T.sub.3 ; 50 ppm                                                            178.37 ± 6.32                                                                      185.12 ± 6.63                                                                      193.80 ± 2.70                                                                      66.22.sup.C                                                                         224.66 ± 3.71                                                                      88.61.sup.B                             (100.00)                                                                              (103.78)                                                                              (108.65)      (125.95)                          5) CD + P-21 - T.sub.3 ;                                                                    182.09 ± 14.03                                                                     194.17 ± 11.74                                                                     198.76 ± 9.57                                                                      66.53.sup.C                                                                         227.82 ± 11.33                                                                     88.02.sup.B                 50 ppm      (100.00)                                                                              (106.63)                                                                              (109.15)      (125.11)                          6) CD + P-25 - T.sub.3 ;                                                                    182.37 ± 11.92                                                                     191.05 ± 8.78                                                                      198.01 ± 6.04                                                                      65.51.sup.C                                                                         233.75 ± 7.49                                                                      90.17.sup.B                 50 ppm      (100.00)                                                                              (104.76)                                                                              (107.48)      (128.17)                          __________________________________________________________________________     .sup.1 Feeding period was 6 weeks. Time of drawing the blood was 0.009 hr     .sup.2 Data expressed as means ± SD;; n = 6 samples of 3 swine per         group; TRF = TocotrienolRich-Fraction.                                        .sup.3 Percentages of increases or decreases in respect to baseline value     .sup.A-C Values not sharing a common superscript letter are different at      < 0.01.                                                                  

                                      TABLE XV                                    __________________________________________________________________________    HDL:LDL CHOLESTEROL CONCENTRATION IN SERUM (mg/100 ml).sup.2                                Experimental Diets                                                                           Percentage                                                                          Control Diet                                                                          Percentage                                       Feeding Period of Control                                                                          Feeding Period                                                                        of Control                         NUTRITIONAL STATE.sup.1                                                                     0-Time                                                                             21 days                                                                           42 days                                                                             Activity                                                                            70 days Activity                           __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                        1:4.47                                                                             1:4.54                                                                             1:3.90                                                                             --    1:3.89  --                                               (100.00).sup.3                                                                     (101.57).sup.3                                                                     (87.25).sup.3                                                                            (87.02).sup.3                              Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        1:8.02                                                                             1:8.38                                                                             1:11.19                                                                            100.00.sup.A                                                                        1:8.72  100.00.sup.A                                     (100.00)                                                                           (104.49)                                                                           (139.53)                                              3) CD + TRF RD-oil;                                                                         1:7.59                                                                             1:7.44                                                                             1:7.89                                                                             70.51.sup.B                                                                         1:8.85  101.41.sup.A                         50 ppm      (100.00)                                                                           (98.02)                                                                            (103.95)                                              4) CD + γ.sub.3 ; 50 ppm                                                              1:8.16                                                                             1:7.33                                                                             1:6.79                                                                             60.58.sup.C                                                                         1:7.37  84.52.sup.B                                      (100.00)                                                                           (89.83)                                                                            (82.84)                                               5) CD + P-21 - T.sub.3 ;                                                                    1:7.93                                                                             1:7.40                                                                             1:6.47                                                                             57.82.sup.C                                                                         1:7.31  83.83.sup.B                          50 ppm      (100.00)                                                                           (93.32)                                                                            (81.59)                                               6) CD + P-25 - T.sub.3 ;                                                                    1:8.34                                                                             1:7.44                                                                             1:6.22                                                                             55.59.sup.D                                                                         1:7.16  82.06.sup.B                          50 ppm      (100.00)                                                                           (89.20)                                                                            (74.58)                                               __________________________________________________________________________     .sup.1 Feeding period was 6 weeks. Time of drawing the blood was 0.009 hr     TRF = TocotrienolRich-Fraction.                                               .sup.2 Data expresssed as means ± SD; n = 6 samples of 3 swine per         group.                                                                        .sup.3 Percentages of increases or decreases in respect to baseline value     A-DValues not sharing a common superscript letter are different at P <        0.01.                                                                    

All tocotrienols tested caused the levels of total serum cholesterol andLDL-cholesterol to decrease by about 25% and 10%, respectively. Inaddition, the HDL-/LDL-cholesterol ratio was reduced by about 20% withall three tocotrienols tested. These reductions were all time dependent.Moreover, the reductions observed after 42 days persisted over the 10weeks following termination of tocotrienol feeding. This result suggeststhat the tocotrienols remain in the blood stream over extended periodsof time. While not wishing to be bound by theory, we believe that thetocotrienols might be transported in association with the LDL- and HDL-lipoproteins. Alternatively, the tocotrienols may also bind to otherproteins.

Example 17

This example was conducted to measure the serum levels of apolipoprotein(a)₁, apolipoprotein B, triglycerides, glucose, insulin and glucagon andthe plasma levels of thromboxane B₂ and platelet factor 4 in the swinedescribed in Example 15.

The samples were prepared as described in Example 12.

The results of these studies are displayed in Tables XVI and XVII.Percentages of increases and decreases are shown in parentheses.

                                      TABLE XVI                                   __________________________________________________________________________                  Concentration In Serum (mg/100 ml)                              NUTRITIONAL STATE.sup.1                                                                      Apo A.sub.1                                                                          Apo B  Triglycerides                                    __________________________________________________________________________    Hypercholesterolemic Swine                                                    1) Control Diet (CD)                                                                         25.69 ± 1.19                                                                      147.77 ± 1.50                                                                     76.56 ± 1.22                                                 (100.00).sup.A                                                                       (100.00).sup.A                                                                       (100.00).sup.A                                   2) CD + TRF RB-oil; 50 ppm                                                                   26.26 ± 1.07                                                                      118.39 ± 1.50                                                                     66.67 ± 1.39                                                 (102.22).sup.A                                                                       (80.12).sup.A                                                                        (106.15).sup.B                                   3) CD + γ - T.sub.3 ; 50 ppm                                                           26.22 ± 1.11                                                                      114.29 ± 1.79                                                                     61.78 ± 1.09                                                 (102.06).sup.A                                                                       (77.34).sup.C                                                                        (80.69).sup.C                                    4) CD + P-21 - T.sub.3 ; 50 ppm                                                              26.72 ± 1.14                                                                      111.78 ± 1.80                                                                     64.44 ± 1.29                                                 (104.01).sup.A                                                                       (75.64).sup.C                                                                        (84.17).sup.B                                    5) CD + P-25 - T.sub.3 ; 50 ppm                                                              26.73 ± 1.22                                                                      106.63 ± 1.72                                                                     62.00 ± 1.19                                                 (104.05).sup.A                                                                       (72.16).sup.D                                                                        (80.98).sup.C                                    __________________________________________________________________________     .sup.1 Feeding period was 6 weeks. Time of drawing the blood was 0.009 hr     .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group; TRF = TocotrienolRich-Fraction.                                        .sup.3 Percentages of increases or decreases in respect to baseline value     .sup.A-D Values not sharing a common superscript letter are different at      < 0.01.                                                                  

                                      TABLE XVII                                  __________________________________________________________________________                                                 Platelet                                       Glucose                                                                              Insulin                                                                              Glucagon                                                                              Thromboxane B.sub.2                                                                    Factor 4                         NUTRITIONAL STATE.sup.1                                                                     (mg/100 ml)                                                                          (μG/ML)                                                                           (pg/ml) (pg/100 ml)                                                                            (mg/100 ml)                      __________________________________________________________________________    Hypercholesterolemic                                                          Swine                                                                         1) Control Diet (CD)                                                                        95.99 ± 1.37.sup.A                                                                10.50 ± 0.12.sup.A                                                                330.41 ± 14.08.sup.A                                                               75.93 ± 1.45.sup.A                                                                  24.12 ± 1.75.sup.A                          (100.00)                                                                             (100.00)                                                                             (100.00)                                                                              (100.00) (100.00)                         2) CD + TRF RB-oil;                                                                         74.45 ± 1.03.sup.B                                                                23.10 ± 1.08.sup.B                                                                295.36 ± 10.45.sup.B                                                               64.55 ± 1.18B                                                                       20.32 ± 1.70.sup.B              50 ppm      (77.56)                                                                              (112.68)                                                                             (89.39) (95.23)  (84.30)                          3) CD + γ - T.sub.3 ; 50 ppm                                                          74.40 ± 1.12.sup.B                                                                21.21 ± 0.89.sup.B                                                                285.41 ± 13.88.sup.B                                                               62.54 ± 1.39.sup.B                                                                  20.65 ± 1.15.sup.B                          (77.51)                                                                              103.46)                                                                              (86.38) (92.25)  (85.28)                          4) CD + P-21 - T.sub.3 ;                                                                    72.63 ± 1.58.sup.B                                                                21.45 ± 0.87.sup.B                                                                278.36 ± 16.42.sup.B                                                               60.48 ± 1.46.sup.B                                                                  19.85 ± 1.27.sup.B,C            50 ppm      (75.66)                                                                              (104.63)                                                                             (84.25) (87.66)  (82.59)                          5) CD + P-25 - T.sub.3 ;                                                                    71.89 ± 1.89.sup.B                                                                20.89 ± 1.79.sup.B                                                                274.41 ± 12.32.sup.B                                                               57.03 ± 1.95.sup.B,C                                                                19.15 ± 1.45.sup.B,C            50 ppm      (74.89)                                                                              (101.90)                                                                             (83.05) (83.02)  (79.36)                          __________________________________________________________________________     .sup.1 Cornsoybean mean control diet or experimental diets were fed to        5month-old swine for 42 days. Then all groups were fasted for 40 hrs. and     refed for 48 days. The time of sacrificing was 9:00 a.m. All tissues were     kept on ice. TRF = TocotrienolRich-Fraction.                                  .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group;                                                                        .sup.3 Percentages of increases or decreases in respect to baseline value     .sup.A-C Values not sharing a common superscript letter are different at      < 0.01.                                                                  

These results show that all the tocotrienols tested reduced levels ofapolipoprotein B, triglycerides, glucose, glucagon and platelet factor 4by about 30%, 20%, 25%, 17% and 21%, respectively. The tocotrienols didnot substantially affect the levels of apolipoprotein (a)₁ and insulin.

Example 18

We next determined the effect of tocotrienols on the total cholesterol,triglyceride and glucose concentrations in various tissues ofhypercholesterolemic swine.

The samples were prepared as described in Example 12.

The results of these studies are displayed in Tables XVIII-XX.Percentages of increases and decreases are shown in parentheses.

                                      TABLE XVIII                                 __________________________________________________________________________    TOTAL CHOLESTEROL CONCENTRATION (mg/100 gm).sup.2                                                               Loin Adipose                                                                            Ham                               NUTRITIONAL STATE.sup.1                                                                     Liver                                                                              Intestine                                                                          Lung Heart                                                                              Muscle                                                                             Tissue                                                                             Muscle                            __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                        151.59 ±                                                                        175.10 ±                                                                        76.45 ±                                                                         60.50 ±                                                                         97.89 ±                                                                         92.86 ±                                                                         86.09 ±                                      6.22 6.61 7.19 3.49 4.14 5.51 2.57                              Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        201.59 ±                                                                        215.49 ±                                                                        101.74 ±                                                                        64.51 ±                                                                         119.62 ±                                                                        109.40 ±                                                                        100.00 ±                                     5.68.sup.A                                                                         5.78.sup.A                                                                         4.34.sup.A                                                                         9.61.sup.A                                                                         3.89.sup.A                                                                         4.00.sup.A                                                                         3.73.sup.A                                      (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                          3) CD + TRF RB-oil;                                                                         172.22 ±                                                                        198.63 ±                                                                        91.12 ±                                                                         58.78 ±                                                                         112.69 ±                                                                        102.25 ±                                                                        93.05 ±                          50 ppm      4.19.sup.B                                                                         3.75.sup.B                                                                         2.28.sup.B                                                                         1.87.sup.B                                                                         2.63.sup.B                                                                         1.94.sup.A                                                                         3.44.sup.B                                      (85.43)                                                                            (92.18)                                                                            (89.56)                                                                            (91.12)                                                                            (94.21)                                                                            (3.46)                                                                             (93.05)                           4) CD + γ - T.sub.3 ; 50 ppm                                                          149.20 ±                                                                        187.06 ±                                                                        83.79 ±                                                                         56.68 ±                                                                         103.48 ±                                                                        94.55 ±                                                                         85.15 ±                                      3.83.sup.C                                                                         4.27.sup.C                                                                         3.24.sup.C                                                                         1.91.sup.B                                                                         2.39.sup.C                                                                         3.03.sup.C                                                                         2.24.sup.C                                      (74.01)                                                                            (86.81)                                                                            (82.36)                                                                            (87.86)                                                                            (86.51)                                                                            (86.43)                                                                            (85.15)                           5) CD + P-21 - T.sub.3                                                                      13.70 ±                                                                         180.59 ±                                                                        75.68 ±                                                                         53.63 ±                                                                         100.58 ±                                                                        89.85 ±                                                                         79.51 ±                          50 ppm      4.55.sup.D                                                                         3.03.sup.C,D                                                                       4.14.sup.D                                                                         2.01.sup.C                                                                         1.71.sup.C                                                                         2.17.sup.B                                                                         3.75.sup.C                                      (67.81)                                                                            (83.80)                                                                            (74.39)                                                                            (83.13)                                                                            (84.09)                                                                            (82.13)                                                                            (79.51)                           6) CD + P-25 - T.sub.3 ;                                                                    125.21 ±                                                                        176.67 ±                                                                        68.5 ±                                                                          51.53 ±                                                                         99.04 ±                                                                         89.54 ±                                                                         69.17 ±                          50 ppm      4.30.sup.E                                                                         5.41.sup.E                                                                         5.32.sup.D                                                                         2.20.sup.C                                                                         3.52.sup.C                                                                         1.98.sup.B                                                                         3.88.sup.D                                      (62.11)                                                                            (81.98)                                                                            (66.98)                                                                            (82.79)                                                                            (81.85)                                                                            (81.85)                                                                            (69.17)                           __________________________________________________________________________     .sup.1 Cornsoybean meal control diet or experimental diets were fed to        5month-old swine for 42 days. Then all groups were fasted for 40 hrs. and     refed for 48 days. The time of sacrificing was 9:00 a.m. All tissues were     kept on ice. TRF = TocotrienolRich-Fraction.                                  .sup.2 Data expressed as means ± SD; n = 6 samples of 3 swine per          group;                                                                        .sup.A-E Values not sharing a common superscript letter are different at      < 0.01.                                                                       Percentages of control activity are given in parenthesis.                

                                      TABLE XIX                                   __________________________________________________________________________    TOTAL TRIGLYCERIDES CONCENTRATION (mg/100 gm).sup.2                                                             Loin Adipose                                                                            Ham                               NUTRITIONAL STATE.sup.1                                                                     Liver                                                                              Intestine                                                                          Lung Heart                                                                              Muscle                                                                             Tissue                                                                             Muscle                            __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                        388.68 ±                                                                        300.24 ±                                                                        163.53 ±                                                                        130.51 ±                                                                        207.55 ±                                                                        294.43 ±                                                                        187.50 ±                                     5.80 5.63 3.57 3.59 5.50 4.99 3.68                              Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        429.36 ±                                                                        357.63 ±                                                                        171.96 ±                                                                        144.22 ±                                                                        219.68 ±                                                                        435.68 ±                                                                        185.83 ±                                     5.02.sup.A                                                                         4.48.sup.A                                                                         4.04.sup.A                                                                         3.29.sup.A                                                                         5.19.sup.A                                                                         3.79.sup.A                                                                         4.05.sup.A                                      (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                          3) CD + TRF RB-oil;                                                                         409.80 ±                                                                        355.93 ±                                                                        166.66 ±                                                                        140.71 ±                                                                        212.27 ±                                                                        421.42 ±                                                                        184.99 ±                         50 ppm      6.48.sup.B                                                                         3.07.sup.A                                                                         2.49.sup.A                                                                         3.35.sup.A                                                                         7.09.sup.B                                                                         3.45.sup.B                                                                         2.40.sup.A                                      (95.44)                                                                            (99.52)                                                                            (96.92)                                                                            (97.57)                                                                            (96.63)                                                                            (96.73)                                                                            (99.55)                           4) CD + γ - T.sub.3 ; 50 ppm                                                          397.51 ±                                                                        351.33 ±                                                                        167.18 ±                                                                        142.72 ±                                                                        201.65 ±                                                                        391.58 ±                                                                        181.99 ±                                     3.88.sup.C                                                                         2.80.sup.A,B                                                                       2.86.sup.A                                                                         1.28.sup.A                                                                         2.01.sup.C                                                                         7.59.sup.C                                                                         1.67.sup.A                                      (92.58)                                                                            (98.24)                                                                            (97.22)                                                                            (98.96)                                                                            (91.79)                                                                            (89.88)                                                                            (97.93)                           5) CD + P-21 - T.sub.3 ;                                                                    386.25 ±                                                                        347.75 ±                                                                        165.47 ±                                                                        142.38 ±                                                                        194.75 ±                                                                        282.82 ±                                                                        182.16 ±                         50 ppm      5.10.sup.D                                                                         4.74.sup.B                                                                         2.26.sup.A                                                                         1.73.sup.A                                                                         1.72.sup.D                                                                         6.86.sup.C                                                                         1.28.sup.A                                      (89.96)                                                                            (97.24)                                                                            (96.22)                                                                            (98.72)                                                                            (88.65)                                                                            (64.91)                                                                            (98.03)                           6) CD + P-25 - T.sub.3 ;                                                                    384.00 ±                                                                        345.37 ±                                                                        163.42 ±                                                                        140.51 ±                                                                        189.18 ±                                                                        259.62 ±                                                                        182.98 ±                         50 ppm      2.98.sup.D                                                                         3.97.sup.B                                                                         2.46.sup.A                                                                         0.99.sup.A                                                                         1.68.sup.E                                                                         7.47.sup.D                                                                         1.76.sup.A                                      (89.44)                                                                            (96.57)                                                                            (95.03)                                                                            (97.43)                                                                            (86.12)                                                                            (59.59)                                                                            (98.44)                           __________________________________________________________________________     .sup.1 Cornsoybean meal control diet or experimental diets were fed to        5month-old swine for 42 days. Then all groups were fasted for 40 hrs. and     refed for 48 days. The time of sacrificing was 9:00 a.m. All tissues were     kept on ice. TRF = TocotrienolRich-Fraction.                                  .sup.2 Data expressed as means ± SD; n = 4 analysis of the samples per     group;                                                                        .sup.A-D Values not sharing a common superscript letter are different at      < 0.01.                                                                       Percentages of control activity are given in parentheses.                

                                      TABLE XX                                    __________________________________________________________________________    TOTAL GLUCOSE CONCENTRATION (mg/100 gm).sup.2                                                                   Loin Adipose                                                                            Ham                               NUTRITIONAL STATE.sup.1                                                                     Liver                                                                              Intestine                                                                          Lung Heart                                                                              Muscle                                                                             Tissue                                                                             Muscle                            __________________________________________________________________________    Normolipemic Swine                                                            1) Control Diet (CD)                                                                        361.05 ±                                                                        66.86 ±                                                                         61.35 ±                                                                         42.86 ±                                                                         65.79 ±                                                                         21.85 ±                                                                         53.82 ±                                      7.79 5.81 6.45 1.91 2.57 5.01 2.60                              Hypercholesterolemic                                                          Swine                                                                         2) Control Diet (CD)                                                                        405.23 ±                                                                        71.97 ±                                                                         66.93 ±                                                                         49.25 ±                                                                         71.24 ±                                                                         18.34 ±                                                                         53.82 ±                                      7.68.sup.A                                                                         2.97.sup.A                                                                         4.40.sup.A                                                                         2.91.sup.A                                                                         1.26.sup.A                                                                         2.60.sup.A                                                                         2.60.sup.A                                      (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                                                                           (100.00)                          3) CD + TRF RB-oil;                                                                         395.54 ±                                                                        64.02 ±                                                                         66.16 ±                                                                         46.80 ±                                                                         64.48 ±                                                                         17.04 ±                                                                         52.10 ±                          50 ppm      3.82.sup.A                                                                         2.80.sup.B                                                                         3.82.sup.A                                                                         2.50.sup.A                                                                         2.70.sup.B                                                                         1.35.sup.A,B                                                                       1.90.sup.A                                      (97.61)                                                                            (88.95)                                                                            (98.85)                                                                            (95.03)                                                                            (90.51)                                                                            (92.01)                                                                            (96.80)                           4) CD + γ - T.sub.3 ; 50 ppm                                                          383.68 ±                                                                        62.50 ±                                                                         63.08 ±                                                                         46.81 ±                                                                         60.30 ±                                                                         15.93 ±                                                                         52.10 ±                                      3.37.sup.B                                                                         2.36.sup.B,C                                                                       3.38.sup.A,B                                                                       3.21.sup.A                                                                         1.95.sup.B                                                                         1.77.sup.A,B                                                                       1.69.sup.A                                      (94.53)                                                                            (86.84)                                                                            (94.25)                                                                            (95.05)                                                                            (84.64)                                                                            (86.86)                                                                            (96.80)                           5) CD + P-21 - T.sub.3 ;                                                                    374.22 ±                                                                        62.50 ±                                                                         61.92 ±                                                                         44.17 ±                                                                         55.45 ±                                                                         16.67 ±                                                                         54.01 ±                          50 ppm      3.19.sup.C                                                                         2.80.sup.B,C                                                                       2.63.sup.B                                                                         3.55.sup.A                                                                         1.88.sup.C                                                                         1.96.sup.A,B                                                                       1.73.sup.A                                      (92.35)                                                                            (86.84)                                                                            (92.51)                                                                            (89.69)                                                                            (77.84)                                                                            (90.89)                                                                            (100.35)                          6) CD + P-25 - T.sub.3 ;                                                                    362.99 ±                                                                        56.06 ±                                                                         60.96 ±                                                                         45.49 ±                                                                         53.01 ±                                                                         14.96 ±                                                                         53.05 ±                          50 ppm      4.31.sup.D                                                                         4.06.sup.C                                                                         5.41.sup.B                                                                         2.78.sup.A                                                                         1.30.sup.C                                                                         1.26.sup.B                                                                         2.61.sup.A                                      (89.58)                                                                            (77.89)                                                                            (91.08)                                                                            (92.37)                                                                            (74.41)                                                                            (81.57)                                                                            (98.57)                           __________________________________________________________________________     .sup.1 Cornsoybean meal control diet or experimental diets were fed to        5month-old swine for 42 days. Then all groups were fasted for 40 hrs. and     refed for 48 days. The time of sacrificing was 9:00 a.m. All tissues were     kept on ice. TRF = TocotrienolRich-Fraction.                                  .sup.2 Data expressed as means ± SD; n = 4 analysis of the sample per      group;                                                                        .sup.3 Percentages of control activity are given in parentheses.              .sup.A-D Values not sharing a common superscript letter are different at      < 0.01.                                                                  

Decreases in total serum cholesterol were found in the each of thetissues tested. Most notably, total cholesterol concentration wasreduced by 37% and 30% with P₂₅ in the liver and ham muscle,respectively. Total triglyceride and glucose concentrations were lessaffected.

Example 19

We next measured the effects of different tocotrienols of rice bran oilon fatty acid compositions in various tissues of hypercholesterolemicswine.

The levels of fatty acids, measured as their esters, were estimatedusing the method described in K. Hirai et al., "Effects of Dietary Fatsand Phytosterol on Serum Fatty Acid Composition and LipoproteinCholesterol in Rats", J. Nutr. Sci. Vitaminol., 30, pp. 101-112 (1984),except that 1 g of each tissue was mixed with 2 ml of saline solution,the homogenized 30 sec by polytron. Following homogenization, the samplewas extracted with 8 ml of hexane by shaking for 20 min., thencentrifuging for 10 min. at 2000 rpm. The hexane layer was Dried at 40°C. and the residue treated with 0.5 ml diazomethane to yield the fattyacid esters.

The identification of each fatty acid ester was established bycomparison against a standard mixture of fatty acid esters (obtainedfrom Sigma Chemical Co.).

The results, as displayed in FIGS. 1-4, demonstrate that thetocotrienols tested were able to lower the total fatty acidconcentration in every tissue tested. The most impressive declines wereseen in the heart (76%), the loin (81%) and the ham muscle (66%). Thegreatest reductions were seen in levels of arachidonic acid. Thisdecrease may be due to an increase in the corticosterone levels. Such anincrease would inhibit the activity of phospholipase A₂, which would inturn decrease the arachidonic acid metabolites. The overall fatty acidreduction is also important because it points towards animmunoregulatory role of these tocotrienols.

Example 20

We next determined the effect of tocotrienols on immune function. Themodel for this study was 6-week old female white leghorn chickens. Eachgroup of 12 chickens was fed an unsupplemented chicken diet or a controldiet supplemented with 50 ppm of a tocotrienol or 100 ppm of acommercially available hypercholesterolemic agent (Geraniol orLovastatin). The samples were prepared as described in Example 11. Thechickens were fed over a period of 4 weeks, then the blood was drawn.The antibody response was determined by injecting the chickens i.p. with0.5 ml of a 5% suspension of sheep red blood cells (RBCs) in PBS (v/v)which were previously washed three times. Total antibody titers to theRBCs were determined using 50 μg of serum and the microhemagglutinationtechnique described in Witlin, "Detection of Antibodies byMicrotitration Techniques", Mycopth Mycol Appl , 33, pp. 241-257 (1967).Relative IgG levels were determined by using 2-mercaptoethanolpreincubation for 30 min. The relative IgM levels were calculated bysubtracting the IgG antibody levels from total antibody levels. Allantibody levels are expressed as the log base 2 of the highest titerthat was needed to hemagglutinate an equal volume of 0.5% RBCsuspension. The results of this example are displayed below in TableXXI.

                  TABLE XXI                                                       ______________________________________                                        Antibody titers in Serum (Log 2)                                                          Total Ab                                                          Nutritional State.sup.1                                                                   titers     IgG        IgM                                         ______________________________________                                        1)  Control     6.30 ± 2.99.sup.A                                                                     1.40 ± 0.55.sup.A                                                                   5.67 ± 3.09.sup.A                          Group (CD)                                                                2)  CD +        8.75 ± 1.66.sup.A                                                                     1.50 ± 0.54.sup.A                                                                   7.75 ± 1.55.sup.A                          TRF-RBO                                                                       50 ppm                                                                    3)  CD + α -                                                                            7.10 ± 1.51.sup.A                                                                     1.00 ± 0.00.sup.A                                                                   6.60 ± 1.24.sup.A                          T.sub.3 50 ppm                                                            4)  CD + γ -                                                                            6.60 ± 1.51.sup.A                                                                     1.28 ± 0.49.sup.A                                                                   5.83 ± 1.53.sup.A                          T.sub.3 50 ppm                                                            5)  CD + δ -                                                                            8.67 ± 1.78.sup.A                                                                     1.00 ± 0.00.sup.A                                                                   8.25 ± 1.96.sup.A                          T.sub.3 50 ppm                                                            6)  CD +        8.67 ± 1.92.sup.A                                                                     1.50 ± 1.18.sup.A                                                                   7.17 ± 2.04.sup.A                          P-21-T.sub.3                                                                  50 ppm                                                                    7)  CD + P-25-T.sub.3                                                                         10.30 ± 2.43 ± 1.40.sup.A                                                                   8.92 ± 2.61.sup.A                          50 ppm      1.16.sup.B                                                    8)  CD + Geraniol                                                                             5.60 ± 2.11.sup.A                                                                     1.00 ± 0.00.sup.A                                                                   5.42 ± 2.02.sup.A                      9)  Lovestatin  6.33 ± 1.44.sup.A                                                                     1.00 ± 0.00.sup.A                                                                   5.90 ± 1.68.sup.A                          100 ppm                                                                   10) Geraniol +  7.17 ± 2.04.sup.A                                                                     1.00 ± 0.00.sup.A                                                                   6.83 ± 1.99.sup.A                          Lovastatin                                                                    50 ppm +                                                                      50 ppm                                                                    ______________________________________                                         .sup.1 Fedding period was 4 weeks. Time of drawing the blood was 0.800 hr     Date expressed as means + SD; n = 12 birds per group.                         .sup.AB Values not sharing a common superscript letter are different at       P<0.01.                                                                  

Every tocotrienol, most notably P₂₅, increased the total antibody titerand the IgM titer as compared to the control. Slight increases were alsoobserved in the IgG levels.

Example 21

We determined the effect of a known tocotrienol, γ-T₃, on the release ofsuperoxide in human peripheral blood neutrophils. Neutrophils are anextracellular source of oxygen free radicals. Once activated, theseneutrophils can attach to endothelial tissue where they release a potenttoxin, superoxide. Superoxide amplifies the inflammatory response andimpairs local circulation of the blood.

The neutrophils we tested were isolated by density centrifugation onFicoll-Hypaque gradients using conventional methods (see E. Serbinova etal., "Free Radical Recycling and Intramembrane Mobility in theAntioxidant Properties of α-Tocopherol and α-Tocotrienol", Free Rad.Bio. and Med., 10, pp 263-75 (1991)). The neutrophils were then placedin a 96-well plate. γ-T₃ and phorbol myrstate acetate were added to thewells at the same time. The secretion of superoxide anion was measuredas the superoxide dismutase-inhibitable reduction of ferricytochrome C.

The results of this study are displayed in FIG. 5.

The amount of superoxide released was reduced from 19.7 nmole (for 5×10⁵cells/hour) in the control to 8.0 and 0 nmole at γ-T₃ concentrations of10⁻⁶ and 10⁻⁵, respectively

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments which utilize the processes and products of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims, rather than by the specificembodiments which have been presented by way of example.

We claim:
 1. A tocotrienol comprising3,4-dihydro-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H-1-benzopyran-6-ol), and having the structure: ##STR10##2. A tocotrienol comprising: ##STR11##
 3. A composition comprising atocotrienol compound resulting from a process comprising the stepsof:stabilizing a biological material by heating; extracting thebiological material with a solvent to obtain an oil; eluting the oilfrom an HPLC colunto under the following conditions: 20 μl sample,μ-Porasil column (Waters column, 10μ, 4 mm×30 cm) using an isocraticsystem of hexane and isopropanol (99.75% :0.25%, v/v) at a flow rate of1.3 ml/min.; and collecting compounds having an HPLC elution time ofgreater than 22 minutes, said compounds being detectable at anexcitation wave length of about 295 nm, and having an emission wavelength of about 330 nm, UV absorption at about 295 nm, and an ability toinhibit the synthesis β-hydroxy-β-methyl glutaryl coenzyme A reductase.4. The tocotrienol compound according to claim 3, wherein said HPLCelution time is selected from the group consisting of about 32 minutes,about 36 minutes, about 45 minutes and about 54 minute.
 5. Thetocotrienol compound according to claim 3 or 4, wherein the biologicalmaterial is a plant source.
 6. The tocotrienol compound according toclaim 5, wherein the plant source is selected from the group consistingof oats, oat bran, oat bran oil, barley, barley bran, barley bran oil,rice, rice bran and rice bran oil.
 7. The tocotrienol compound accordingto claim 6, wherein the plant source is rice bran or rice bran oil.
 8. Afoodstuff comprising the tocotrienol compound according to any one ofclaim 1 and 2, or mixtures thereof.
 9. A foodstuff comprising atocotrienol compound, or mixtures thereof, said tocotrienol compoundbeing selected fromthe tocotrienol compound according to claim 3 or 4.10. The foodstuff according to claim 8, further comprising a compoundselected from the group consisting of α-tocotrienol, β-tocotrienol,γ-tocotrienol, δ-tocotrienol, and3.4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H-1-benzopyran-6-ol.
 11. The foodstuff according to claim9, further comprising a compound selected from the group consisting ofα-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, and3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H-1-benzopyran-6-ol.
 12. A dietary supplement comprisingthe tocotrienol compound according to any one of claims 1 and 2, ormixtures thereof.
 13. A dietary supplement comprising a tocotrienolcompound, or mixtures thereof, said tocotrienol compound being selectedfromthe tocotrienol compound according to claim 3 or
 4. 14. The dietarysupplement according to claim 13, further comprising a compound selectedfrom the group consisting of α-tocotrienol, β-tocotrienol,γ-tocotrienol, δ-tocotrienol, and3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H-1-benzopyran-6-ol.
 15. The dietary supplement accordingto claim 13, further comprising a compound selected from the groupconsisting of α-tocotrienol, β-tocotrienol, γ-tocotrienol,δ-tocotrienol, and3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H- 1-benzopyran-6-ol.
 16. A pharmaceutical compositioncomprising the tocotrienol compound according to any one of claims 1 and2, or mixtures thereof, and a pharmaceutical carrier.
 17. Apharmaceutical composition comprising a tocotrienol compound, ormixtures thereof, and a pharmaceutical carrier, said tocotrienolcompound being selected from the group consisting of the tocotrienolcompounds according to any one of claims 3 and
 4. 18. The pharmaceuticalcomposition according to claim 16, further comprising a compoundselected from the group consisting of α-tocotrienol, β-tocotrienol,γ-tocotrienol, δ-tocotrienol, and3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H-1-benzopyran-6ol.
 19. The pharmaceutical compositionaccording to claim 17, further comprising a compound selected from thegroup consisting of α-tocotrienol, β-tocotrienol, γ-tocotrienol,δ-tocotrienol, and3,4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3'(E), 7'(E),11'-trienyl)-2H-1-benzopyran-6ol.
 20. A method for treating orpreventing hypocholesterolemic-related diseases, thrombotic diseases,atherogenic diseases, inflammation, immunodeficiencies, fever, edema,diabetes mellitus, pain, septic shock, chronic fatigue syndrome orfunclio laesa, to decrease lipoprotein (a) concentration in the blood orto increase feed conversion efficiency in a patient, comprising the stepof administering to said patient a pharmaceutically effective amount ofthe pharmaceutical composition according to claim
 16. 21. A method fortreating hypocholesterolemic-related diseases, thrombotic diseases,atherogenic diseases, inflammation, immunodeficiencies, fever, edema,diabetes mellitus, pain, septic shock, chronic fatigue syndrome orfunctio laesa, to decrease lipoprotein (a) concentration in the blood orto increase feed conversion efficiency in a patient, comprising the stepof administering to said patient a pharmaceutically effective amount ofthe pharmaceutical composition according to claim
 17. 22. A method fordecreasing the total serum cholesterol and LDL-cholesterol in a human oranimal, comprising the step of administering to said human or animal acholesterol-lowering effective amount of the foodstuff according toclaim
 8. 23. A method for decreasing the total serum cholesterol andLDL-cholesterol in a human or animal, comprising the step ofadministering to said human or animal a cholesterol-lowering effectiveamount of the foodstuff according to claim
 9. 24. A method for deceasingthe total serum cholesterol and LDL-cholesterol in a human or animal,comprising the step of administering to said human or animal acholesterol-lowering effective amount of the dietary supplementaccording to claim
 12. 25. A method for decreasing the total serumcholesterol and LDL-cholesterol in a human or animal, comprising thestep of administering to said human or animal a cholesterol-loweringeffective amount of the dietary supplement according to claim
 13. 26. Amethod for increasing the HDL/LDL cholesterol ratio in a human oranimal, comprising the step of administering to said human or animal acholesterol-lowering effective amount of the foodstuff according toclaim
 8. 27. A method for increasing the HDL/LDL cholesterol ratio in ahuman or animal, comprising the step of administering to said human oranimal a cholesterol-lowering effective amount of the foodstuffaccording to claim
 9. 28. A method for decreasing the HDL/LDLcholesterol ratio in a human or animal, comprising the step ofadministering to said human or animal a cholesterol-lowering effectiveamount of the dietary supplement according to claim
 12. 29. A method fordecreasing the HDL/LDL cholesterol ratio in a human or animal,comprising the step of administering to said human or animal acholesterol-lowering effective amount of the dietary supplementaccording to claim
 13. 30. A method for enhancing the yield oftocotrienol compounds as defined in any one of claims 1 and 2, whereinsaid tocotrienols are obtained from a tocotrienol-rich fraction of abiological source, said method comprising the steps of:(a) dissolvingthe tocotrienol rich fraction from a biological source in a solvent; (b)contacting the dissolved tocotrienol rich fraction with an amine column;and (c) eluting the tocotrienols with a solvent that does notsubstantially elute impurities.
 31. A method for enhancing the yield oftocotrienol compounds as defined in any one of claims 1 and 2 whereinsaid tocotrienols are obtained from a biological source, said methodcomprising the steps of:(a) extracting the biological source with anorganic solvent, (b) heating the extracted source, and (c) extractingthe heated biological source.
 32. The method according to claim 31,wherein the heating of step (b) is accomplished with microwave heat. 33.The method according to claim 30, wherein the biological source is aplant source.
 34. The method according to claim 31, wherein the plantsource is selected from the group consisting of oats, peanuts, pinenuts, oat bran, oat bran oil, barley, barley bran, barley bran oil,rice, rice bran and rice bran oil.
 35. The method according to claim 34,wherein the plant source is rice bran or rice bran oil.
 36. The methodaccording to claim 30, wherein the solvent of step (a) is hexane. 37.The method according to claim 30, wherein the amine column of step (b)is a Bond Elute amine or cyano column.
 38. The method according to claim30, wherein the solvent of step (c) is a gradient system of 0.5%-3%isopropanol in hexane.
 39. The method according to claim 31, wherein thebiological source is a plant source.